Proaerolysin containing protease activation sequences and methods of use for treatment of prostate cancer

ABSTRACT

Disclosed herein are modified proaerolysin (PA) peptide. In some examples, the proteins include a prostate-specific protease cleavage site and can further include a prostate-tissue-specific binding domain which functionally replaces the native PA binding domain. In other examples, the proteins include a furin cleavage site and a prostate tissue-specific binding domain which functionally replaces the native PA binding domain. Methods of using such peptides to treat prostate cancer are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/487,115filed Feb. 18, 2004 (U.S. Pat. No. ______), which is the U.S. NationalStage of International Application No. PCT/US02/27061, filed Aug. 23,2002 (published in English under PCT Article 21(2)), which in turnclaims the benefit of priority to U.S. Provisional Application No.60/314,613, filed Aug. 24, 2001, both applications herein incorporatedby reference in their entirety.

FIELD

This application relates to novel variant proaerolysin (PA) proteins andmethods of their use to treat localized and metastatic prostate cancer.

BACKGROUND

One of every three cancers diagnosed in American males is of prostaticorigin, making prostate cancer the most commonly diagnosed malignancy inmales in the United States (Berges et al. Clin. Cancer Res. 1:473-480,1995). The incidence of prostate cancer in the U.S. has not beendecreased by changes in lifestyle; in fact, the incidence rate ofclinical prostate cancer has increased steadily since the 1930's (Pinskiet al. Cancer Res. 61:6372-6, 2001). Prostate cancer incidence increaseswith age more rapidly than any other type of cancer; less than 1% ofprostate cancers are diagnosed in men less than 50 years of age (Furuyaet al. Cancer Res. 54:6167-75, 1994). Thus, as the life expectancy ofthe male population increases over time, the incidence of clinicalprostate cancer will also increase (Furuya et al. Cancer Res.54:6167-75, 1994).

Currently there is no treatment that significantly prolongs survival inmen with metastatic prostate cancer (Khan and Denmeade. Prostate45:80-83, 2000). Medical castration with oral estrogen (androgenablation) was the first effective systemic therapy for cancer, andremains the most generally useful prostate cancer therapy. Althoughandrogen ablation therapy has a substantial palliative benefit, it haslittle impact on overall survival (Berges et al. Clin. Cancer Res.1:473-480, 1995). This therapy eventually fails because the metastaticprostate cancer within an individual patient is heterogeneously composedof androgen-dependent and androgen-independent cancer cells (Christensenet al. Bioorg. Medicinal Chem. 7:1273-80, 1999). Following androgenablation, androgen-dependent cells within these tumors stopproliferating and activate a cellular suicide pathway termed programmedcell death (PCD) or apoptosis. Because of the elimination of thissubgroup of androgen-dependent cells, the majority of men withmetastatic prostate cancers have a beneficial response toandrogen-deprivation therapy. However, all patients eventually relapseto a state unresponsive to further anti-androgen therapy, no matter howcompletely given, due to the presence of androgen-independent prostatecancer cells within the metastatic sites. Unfortunately, the disease isuniformly fatal at this point because currently there is no therapy thateffectively eliminates androgen-independent prostate cancer cells (Khanand Denmeade. Prostate 45:80-83, 2000).

Several alternative approaches to the treatment of prostate cancer havebeen proposed. One has been to develop methods to aggressively screenfor local disease while it is still in the prostate and thus potentiallytreatable by definitive local therapy. Localized cancers are oftenmoderately differentiated and smaller in volume. During the last severaldecades, there have been improvements to the surgical andradiotherapeutic management of localized prostate cancer. Theseimprovements have culminated over the last several years in the deathrate of prostate cancer decreasing for the first time in fifty years.

However, while this advance increased the cure rate, there are still alarge number of men who are not cured by local therapies and eventuallydie from metastatic disease. This clinical reality has led to thedevelopment of non-hormonal treatments for metastatic prostate cancer.Standard anti-proliferative chemotherapeutic agents have not beensuccessful as treatment for prostate cancer. These types of agents maybe ineffective against androgen-independent prostatic cancers becausethese cancers have a remarkably low rate of proliferation when comparedto other tumor types and many normal tissues such as skin,gastrointestinal tract and bone marrow. For example, the growth fractionin 117 metastatic sites of prostate cancer obtained from 11 androgenablation failing patients at “warm” autopsy was 7.1±0.8%. (Pinski et al.Cancer Res. 61:6372-6, 2001). This low proliferative rate may explainthe relative unresponsiveness of prostate cancer cells in humans tostandard anti-proliferative chemotherapy, while highly proliferativeandrogen independent prostate cancer cell lines remain exquisitelysensitive to PCD induction in vitro.

Several strategies have been proposed for treatment ofslowly-proliferating prostate cancers. One approach is to identifyspecific signaling pathways to which prostate cancer cells, duringmalignant transformation, acquire a unique dependence for survival. Onceidentified, small molecule or biological inhibitors of these pathwayscan be developed as therapeutics. An example of this approach is the useof small molecule or monoclonal antibody inhibitors of the Her2/neu orEGF receptor pathways. Another method is to inhibit a ubiquitousintracellular protein whose function is mandatory for survival of allcell types. This approach would overcome the problem of heterogeneityand “resistance” as all cancer cells within a tumor could be killed viathis approach. However, the cytotoxicity would not be cell-type specificand administration of such a general toxin would be associated withsignificant systemic toxicity. Therefore, there is a need for a methodfor targeting cytotoxins directly to sites of prostate cancer.

Another strategy for treatment of slowly proliferating prostate cancersis to deliver cytotoxins that kill cells not through induction ofapoptosis following inhibition of critical signaling or metabolicpathways but rather through non-specific cytolysis via disruption of theplasma membrane. Many cytolytic toxins have been described (Lesieur etal. Mol. Membr. Biol. 14:45064, 1997). These cytolytic toxins are oftenof bacterial origin, and, in general, are beta-sheet proteins thatoligomerize in the plasma membrane to produce well-characterized poresthat, once formed, lead to rapid cytolytic cell death (Rossjohn et al.J. Struct. Biol. 121:92-100, 1998). These toxins are also non-specificin their ability to kill cells, and therefore can not be administered astherapy without significant toxicity. Therefore, there is a need foragents to treat prostate cancer, which are predominantly cytotoxic toprostate cancer cells.

SUMMARY

Disclosed herein are variant proaerolysin (PA) molecules and methods oftheir use for treatment of localized and metastatic prostate cancers,such as slowly-proliferating prostate cancers.

In one example, a variant PA molecule includes a prostate-specificprotease cleavage site, such as a prostate-specific antigen (PSA)-,prostate specific membrane antigen (PSMA)-, or human glandularkallikrein 2 (HK2)-specific cleavage site that functionally replaces thenative PA furin cleavage site. In this way, administration of thedisclosed variant PA molecules to a subject having prostate cancerresults in activation of the disclosed variant PA molecules in thepresence of the prostate-specific protease, and lysis of the cells, suchas prostate cancer cells. In some examples, variant PA molecules alsoinclude a prostate tissue specific binding domain, to enhance targetingto cancer cells.

In another example, a variant PA molecule includes a furin cleavagesite, and a prostate tissue specific binding domain which functionallyreplaces the native PA binding domain, to assist in targeting toprostate cancer cells.

Methods are disclosed for treatment of localized and metastatic prostatecancers using the disclosed variant PA molecules. In addition, methodsare disclosed for stimulating a subject's immune system to enhancetreatment of localized and metastatic prostate cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of proaerolysin domains (not drawn to scale) andshows the result of activation by furin.

FIG. 2 is a bar graph showing the results of a hemolysis assay in whichPSA-PA1 is preincubated with human plasma or human plasma spiked withenzymatically active PSA (10,000 ng/ml).

FIG. 3 is a graph comparing the in vitro toxicity of severalproaerolysin variants which include a PSA cleavage site in place of thenative furin site, to wild-type proaerolysin.

FIG. 4 is a bar graph comparing the specificity of PSA-PA1 inPSA-producing (LNCaP), and non-PSA producing (SN12C) tumors, in vivo.

FIGS. 5A-5M are schematic drawings (not to scale) showing how aproaerolysin sequence can be altered to generate several differentvariant PA molecules. The “*” symbol represents one or more pointmutations, and/or one or more deletions which decrease PA binding domainfunction (i.e. the ability to concentrate in a cell membrane).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NOS: 1 and 2 show a wild-type proaerolysin cDNA and proteinsequence, respectively.

SEQ ID NOS: 3 and 4 show the PSA-PA1 cDNA and protein sequence,respectively, wherein the furin site of proaerolysin has been replacedwith a PSA cleavage site.

SEQ ID NOS: 5 and 15-21 are PSA cleavage sites found in humansemenogelin I and II proteins.

SEQ ID NOS: 6 and 7 show the PSA-1K cDNA and protein sequence,respectively, wherein the furin site of proaerolysin has been replacedwith a PSA cleavage site.

SEQ ID NO: 8, 11, and 14-21 are alternative PSA cleavage sites.

SEQ ID NOS: 9 and 10 show the PSA-PA2 cDNA and protein sequence,respectively, wherein the furin site of proaerolysin has been replacedwith a PSA cleavage site.

SEQ ID NOS: 12 and 13 show the PSA-PA3 cDNA and protein sequence,respectively, wherein the furin site of proaerolysin has been replacedwith a PSA cleavage site.

SEQ ID NO: 22 is a native luteinizing hormone releasing hormone (LHRH)protein sequence.

SEQ ID NO: 23 is a modified LHRH protein sequence.

SEQ ID NO: 24 is a protein sequence of a variant PA peptide, wherein thefurin site of PA has been replaced with a PSA cleavage site, and whereinthe native binding domain of PA is deleted and replaced with SEQ ID NO:23.

SEQ ID NO: 25 is a protein sequence of a variant PA peptide, wherein thefurin site of PA is retained, and the native binding domain of PA hasbeen deleted and replaced with SEQ ID NO: 23.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein and inthe appended claims, the singular forms “a” or “an” or “the” includeplural references unless the context clearly dictates otherwise. Forexample, reference to “a variant PA molecule” includes a plurality ofsuch molecules and reference to “the antibody” includes reference to oneor more antibodies and equivalents thereof known to those skilled in theart, and so forth.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs.

Aerolysin: A channel-forming toxin produced as an inactive protoxincalled proaerolysin (PA) (wild-type PA is shown in SEQ ID NOS: 1 and 2).The PA protein contains many discrete functionalities that include abinding domain (approximately amino acids 1-83 of SEQ ID NO: 2), a toxindomain (approximately amino acids 84-426 of SEQ ID NO: 2), and aC-terminal inhibitory peptide domain (approximately amino acids 427-470of SEQ ID NO: 2) that contains a protease activation site (amino acids427-432 of SEQ ID NO: 2).

The binding domain recognizes and binds to glycophosphatidylinositol(GPI) membrane anchors, such as are found in Thy-1 on T lymphocytes, thePIGA gene product found in erythrocyte membranes and Prostate Stem CellAntigen (PSCA). Most mammalian cells express GPI anchored proteins ontheir surfaces. The activation or proteolysis site within proaerolysinis a six amino acid sequence that is recognized as a proteolyticsubstrate by the furin family of proteases. PA is activated uponhydrolysis of a C-terminal inhibitory segment by furin (FIG. 1).Activated aerolysin binds to GPI-anchored proteins in the cell membraneand forms a heptamer that inserts into the membrane producingwell-defined channels of ˜17 Å. Channel formation leads to rapid celldeath via necrosis. Wild-type aerolysin is toxic to mammalian cells,including erythrocytes, for example at 1 nanomolar or less.

Animal: Living multicellular vertebrate organisms, a category whichincludes, for example, mammals and birds.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (e.g., IgG) includes four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. However, the antigen-binding function of an antibodycan be performed by fragments of a naturally occurring antibody. Thus,these antigen-binding fragments are also intended to be designated bythe term antibody. Examples of binding fragments encompassed within theterm antibody include (i) an Fab fragment consisting of the VL, VH, CLand CH1 domains; (ii) an Fd fragment consisting of the VH and CH1domains; (iii) an Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (iv) a dAb fragment (Ward et al., Nature341:544-6, 1989) which consists of a VH domain; (v) an isolatedcomplimentarity determining region (CDR); and (vi) an F(ab′)2 fragment,a bivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region. Furthermore, although the two domains of theFv fragment are coded for by separate genes, a synthetic linker can bemade that enables them to be made as a single protein chain (known assingle chain Fv (scFv); Bird et al. Science 242:423-6, 1988; and Hustonet al., Proc. Natl. Acad. Sci. 85:5879-83, 1988) by recombinant methods.Such single chain antibodies are also included. In one embodiment, anantibody includes camelized antibodies (for example see Tanha et al., J.Biol. Chem. 276:24774-80, 2001).

In one example, antibody fragments are capable of crosslinking theirtarget antigen, e.g., bivalent fragments such as F(ab′)2 fragments.Alternatively, an antibody fragment which does not itself crosslink itstarget antigen (e.g., a Fab fragment) can be used in conjunction with asecondary antibody which serves to crosslink the antibody fragment,thereby crosslinking the target antigen. Antibodies can be fragmentedusing conventional techniques and the fragments screened for utility inthe same manner as described for whole antibodies. An antibody isfurther intended to include bispecific and chimeric molecules thatspecifically bind the target antigen.

“Specifically binds” refers to the ability of individual antibodies tospecifically immunoreact with an antigen. The binding is a non-randombinding reaction between an antibody molecule and an antigenicdeterminant of the T cell surface molecule. The desired bindingspecificity is typically determined from the reference point of theability of the antibody to differentially bind the T cell surfacemolecule and an unrelated antigen, and therefore distinguish between twodifferent antigens, particularly where the two antigens have uniqueepitopes. An antibody that specifically binds to a particular epitope isreferred to as a “specific antibody”.

Cancer: Malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increase rate of growth, invasion ofsurrounding tissue, and is capable of metastasis.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences which determinetranscription. cDNA can be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chemical synthesis: An artificial means by which one can make a proteinor peptide. A synthetic protein or peptide is one made by suchartificial means.

Chemotherapy: In cancer treatment, chemotherapy refers to theadministration of one or a combination of compounds to kill or slow thereproduction of rapidly multiplying cells. Chemotherapeutic agentsinclude those known by those skilled in the art, including, but notlimited to: 5-fluorouracil (5-FU), azathioprine, cyclophosphamide,antimetabolites (such as Fludarabine), antineoplastics (such asEtoposide, Doxorubicin, methotrexate, and Vincristine), carboplatin,cis-platinum and the taxanes, such as taxol and taxotere. Such agentscan be co-administered with the disclosed variant PA molecules to asubject. Alternatively or in addition, chemotherapeutic agents can beadministered prior to and/or subsequent to administration of thedisclosed variant PA molecules to a subject. In one example,chemotherapeutic agents are co-administered with hormonal and radiationtherapy, along with the disclosed variant PA molecules, for treatment ofa localized prostate carcinoma.

Conservative substitution: One or more amino acid substitutions (forexample 2, 5 or 10 residues) for amino acid residues having similarbiochemical properties. Typically, conservative substitutions havelittle to no impact on the activity of a resulting polypeptide. Forexample, ideally, a modified PA peptide including one or moreconservative substitutions retains proaerolysin activity. A polypeptidecan be produced to contain one or more conservative substitutions bymanipulating the nucleotide sequence that encodes that polypeptideusing, for example, standard procedures such as site-directedmutagenesis or PCR.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Examples of amino acids which may be substituted for anoriginal amino acid in a protein and which are regarded as conservativesubstitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glufor Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Glnfor His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leuor Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyrfor Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

Permissive substitutions are non-conservative amino acid substitutions,but also do not significantly alter proaerolysin activity. An example issubstitution of Cys for Ala at position 300 of SEQ ID NO: 2 or 4.

Further information about conservative substitutions can be found in,among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988), WO 00/67796 (Curd et al.) and in standard textbooks of geneticsand molecular biology.

In one example, such variants can be readily selected for additionaltesting by performing an assay (such as those described in Examples 2-5)to determine if the variant retains variant PA activity.

Comprises: A term that means “including.” For example, “comprising A orB” means including A or B, or both A and B, unless clearly indicatedotherwise.

Deletion: The removal of a sequence of a nucleic acid, for example DNA,the regions on either side being joined together.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

Enhance: To improve the quality, amount, or strength of something. Inone embodiment, a therapy enhances the ability of a subject to reducetumors, such as a prostate carcinoma, in the subject if the subject ismore effective at fighting tumors. In another embodiment, a therapyenhances the ability of an agent to reduce tumors, such as a prostatecarcinoma, in a subject if the agent is more effective at reducingtumors. Such enhancement can be measured using the methods disclosedherein, for example determining the decrease in tumor volume (seeExample 5).

Functional Deletion: A mutation, partial or complete deletion,insertion, or other variation made to a gene sequence which renders thatpart of the gene sequence non-functional.

For example, functional deletion of a PA binding domain results in adecrease in the ability of PA to bind to and concentrate in the cellmembrane. This functional deletion can be reversed by inserting anotherfunctional binding domain into proaerolysin, such as a prostate-specificbinding domain, for example, an LHRH peptide.

Examples of methods that can be used to functionally delete aproaerolysin binding domain, include, but are not limited to: deletionof about amino acids 1-83 of SEQ ID NO: 2 or fragments thereof, such asabout amino acids 45-66 of SEQ ID NO: 2, or inserting one or more of thefollowing mutations into a variant proaerolysin sequence W45A, 147E,M57A, Y61A, K66Q (amino acid numbers refer to SEQ ID NO: 2) (forexample, see Mackenzie et al. J. Biol. Chem. 274: 22604-22609, 1999).

In another example, functional deletion of a native PA furin cleavagesite results in a decrease in the ability of PA to be cleaved andactivated by furin, when compared to a wild-type PA molecule.

Immobilized: Bound to a surface, such as a solid surface. A solidsurface can be polymeric, such as polystyrene or polypropylene. In oneembodiment, the solid surface is in the form of a bead. In anotherembodiment, the surface includes a modified PA toxin, and in someexamples further includes one or more prostate-specific binding ligands,such as LHRH peptide, PSMA antibody, and PSMA single chain antibody.Ideally, the modified PA toxin is liberated from the bead once the beadreaches the prostate cell target. Methods of immobilizing peptides on asolid surface can be found in WO 94/29436, and U.S. Pat. No. 5,858,358.

Examples of how the molecules can be attached to the bead include, butare not limited to: PA toxin-PSA cleavage site-bead-prostate bindingligand; or prostate binding ligand-bead-PSA site-PA toxin-PSA cleavagesite-inhibitor.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule or protein) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs (i.e. other chromosomal andextrachromosomal DNA and RNA). Nucleic acids and proteins that have been“isolated” include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids and proteins.

An isolated cell is one which has been substantially separated orpurified away from other biological components of the organism in whichthe cell naturally occurs.

Malignant: Cells which have the properties of anaplasia invasion andmetastasis.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects. Examplesof mammals include, but are not limited to: humans, pigs, cows, goats,cats, dogs, rabbits and mice.

Neoplasm: Abnormal growth of cells.

Normal Cell: Non-tumor cell, non-malignant, uninfected cell.

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotide bases in length, for example a polynucleotide (such as DNA orRNA) which is at least about 6 nucleotides, for example at least 15, 50,100 or 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Polynucleotide: A linear nucleic acid sequence of any length. Therefore,a polynucleotide includes molecules which are at least 15, 50, 100, 200,400, 500, 1000, 1100, or 1200 (oligonucleotides) and also nucleotides aslong as a full-length cDNA or chromosome.

Proaerolysin: The inactive protoxin of aerolysin. The cDNA and proteinof a wild-type or native proaerolysin are shown in SEQ ID NOS: 1 and 2,respectively.

In one example, a variant or modified proaerolysin molecule includes aprostate-specific protease cleavage site, such as a PSA-specificcleavage site, which permits activation of the variant PA in thepresence of a prostate-specific protease such as PSA, PMSA, or HK2 (forexample, see FIGS. 5C-I). In one example, a prostate-specific proteasecleavage site is inserted into the native furin cleavage site of PA,such that PA is activated in the presence of a prostate-specificprotease, but not furin (for example see FIGS. 5D-I). Alternatively, thefurin cleavage site can be functionally deleted using mutagenesis of thesix amino acid sequence, and insertion of a prostate-specific proteasecleavage sequence (for example, see FIG. 5C). In another example, avariant PA molecule further includes deletion or substitution of one ormore, such as at least two, of the native PA amino acids. In yet anotherexample a variant PA molecule further includes another molecule (such asan antibody or peptide) linked or added to (or within) the variant PAmolecule. In another example, a variant PA molecule includes aprostate-tissue specific binding domain.

In another example, a variant PA molecule further includes afunctionally deleted binding domain (about amino acids 1-83 of SEQ IDNO: 2). Functional deletions can be made using any method known in theart, such as deletions, insertions, mutations, or substitutions.Examples include, but are not limited to deleting the entire bindingdomain (or portions thereof, (for example, see FIGS. 5G-I), orintroduction of point mutations (such as those described above, (forexample, see FIGS. 5D-F), which result in a binding domain withdecreased function. For example, a PA molecule which has a functionallydeleted binding domain (and no binding sequence substituted therefor),will have a decreased ability to accumulate in a cell membrane, andtherefore lyse cells at a slower rate than a wild-type PA sequence (forexample, see FIG. 5D). Also disclosed are variant PA molecules in whichthe native binding domain is functionally deleted and replaced with aprostate-tissue specific binding domain as described below (for example,see FIGS. 5E-I).

In another example, a variant or modified PA molecule includes a furincleavage site, and a functionally deleted binding domain which isreplaced with a prostate-tissue specific binding domain (for example,see FIGS. 5J-M). Such variant PA molecules are targeted to prostatecells via the prostate-tissue specific binding domain, and activated inthe presence of furin.

Particular non-limiting examples of variant PSA proteins are shown inSEQ ID NOS: 4, 7, 10, 13, 24, and 25.

Modified PA activity is the activity of an agent in which the lysis ofcells is affected. Cells include, but are not limited toprostate-specific protease secreting cells, such as PSA-secreting cells,such as prostate cancer cells, such as slow-proliferating prostatecancer cells. Agents include, but are not limited to, modified PAproteins, nucleic acids, specific binding agents, including variants,mutants, polymorphisms, fusions, and fragments thereof, disclosedherein. In one example, modified PA activity is said to be enhanced whenmodified PA proteins or nucleic acids, when contacted with aPSA-secreting cell (such as a prostate cancer cell), promote lysis anddeath of the cell, for example by at least 10%, or for example by atleast 25%, 50%, 100%, 200% or even 500%, when compared to lysis of anon-PSA producing cell. In other examples, modified PA activity is saidto be enhanced when modified PA proteins and nucleic acids, whencontacted with a tumor, decrease tumor cell volume, such as a prostatetumor, for example by at least 10% for example by at least 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even 100% (completeelimination of the tumor).

Assays which can be used to determine if an agent has modified PAactivity are described herein, for example in Examples 2-5 and 9. Forexample, a modified PA peptide can be assessed for its ability tospecifically lyse PSA-producing cells (Examples 2 and 5), be stable inhuman plasma (Example 3), be an efficient substrate for the enzymaticactivity of PSA (Example 9). Functional protein activity could bedetected by the preferential lysis of PSA-producing cells versusnon-PSA-producing cells, decreasing prostate tumor volume, having adecreased toxicity when compared to wild-type PA, and having anincreased stability in blood when compared to wild-type PA.

Similar assays can be used to determine if any modified PA agentdisclosed herein can decrease tumor volume (such as a prostate tumor)and specifically lyse PSA-producing cells. For example, the modified PApeptides shown in SEQ ID NOS: 4, 24, and 25, are expected to decreaseprostate tumor volume. Any of these assays can be modified by using invivo expression of a modified PA gene, and variants, fusions, andfragments thereof, instead of administration of purified proteins.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription.

Prostate-specific promoter: A promoter responsive to testosterone andother androgens, which therefore promotes gene expression in prostatecells. Examples include, but are not limited to the probasin promoter;the prostate specific antigen (PSA) promoter; the prostate specificmembrane antigen (PSMA) promoter; and the human glandular kallikrein 2(HK2) promoter.

Prostate-specific protease cleavage site: A sequence of amino acidswhich is recognized and specifically and efficiently hydrolyzed(cleaved) by a prostate-specific protease. Examples include, but are notlimited to a PSA-specific cleavage site, a PSMA-specific cleavage siteand an HK2-specific cleavage site.

A PSA-specific cleavage site is a sequence of amino acids which isrecognized and specifically and efficiently hydrolyzed (cleaved) byprostate specific antigen (PSA). Such peptide sequences can beintroduced into other molecules, such as PA, to produce prodrugs thatare activated by PSA. Upon activation of the modified PA by PSA, PA isactivated and can exert its cytotoxicity. Examples of PSA-specificcleavage sites, include, but are not limited to are those shown in SEQID NOS: 5, 8, 11, and 14-21, those disclosed in U.S. Pat. Nos. 5,866,679to DeFeo-Jones et al., 5,948,750 to Garsky et al., 5,998,362 to Feng etal., 6,265,540 to Isaacs et al., 6,368,598 to D'Amico et al., and6,391,305 to Feng et al., (all herein incorporated by reference).

Particular examples of PSMA-specific cleavage sites can be found inWO/0243773 to Isaacs and Denmeade (herein incorporated by reference).Particular examples of HK2-specific cleavage sites are disclosed inWO/0109165 to Denmeade et al. (herein incorporated by reference).

Prostate tissue-specific binding domain: A molecule, such as a peptideligand, toxin, or antibody, which has a higher specificity for prostatecells than for other cell types. In one example, a prostate tissuespecific binding domain has a lower K_(D) in prostate tissue or cellsthan in other cell types, (i.e. binds selectively to prostate tissues ascompared to other normal tissues of the subject), for example at least a10-fold lower K_(D), such as an at least 20-, 50-, 75-, 100- or even200-fold lower K_(D). Such sequences can be used to target an agent,such as a variant PA molecule, to the prostate. Examples include, butare not limited to: antibodies which recognize proteins that arerelatively prostate-specific such as PSA, PSMA, hK2, prostasin, andhepsin; ligands which have prostate-selective receptors such as naturaland synthetic luteinizing hormone releasing hormone (LHRH); andendothelin (binding to cognate endothelin receptor).

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a substantiallypurified protein or nucleic acid preparation (such as the modified PAtoxins disclosed herein) is one in which the protein or nucleic acidreferred to is more pure than the protein in its natural environmentwithin a cell or within a production reaction chamber (as appropriate).For example, a preparation of a modified PA protein is purified if theprotein represents at least 50%, for example at least 70%, of the totalprotein content of the preparation. Methods for purification of proteinsand nucleic acids are well known in the art. Examples of methods thatcan be used to purify a protein, such as a modified PA, include, but arenot limited to the methods disclosed in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 17).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. A recombinantprotein is one that results from expressing a recombinant nucleic acidencoding the protein.

Sample: Biological samples containing genomic DNA, cDNA, RNA, or proteinobtained from the cells of a subject, such as those present inperipheral blood, urine, saliva, semen, tissue biopsy, surgicalspecimen, fine needle aspirates, amniocentesis samples and autopsymaterial. In one example, a sample includes prostate cancer cellsobtained from a subject.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequence will show increasingpercentage identities when assessed by this method, such as at least70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity. When lessthan the entire sequence is being compared for sequence identity,homologs will typically possess at least 75% sequence identity overshort windows of 10-20 amino acids, and can possess sequence identitiesof at least 85%, 90%, 95% or 98% depending on their identity to thereference sequence. Methods for determining sequence identity over suchshort windows are described at the NCBI web site.

Protein homologs are typically characterized by possession of at least70%, such as at least 75%, 80%, 85%, 90%, 95% or even 98% sequenceidentity, counted over the full-length alignment with the amino acidsequence using the NCBI Basic Blast 2.0, gapped blastp with databasessuch as the nr or swissprot database. Queries searched with the blastnprogram are filtered with DUST (Hancock and Armstrong, 1994, Comput.Appl. Biosci. 10:67-70). Other programs use SEG.

One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is possible that stronglysignificant homologs could be obtained that fall outside the rangesprovided. Provided herein are the peptide homologs described above, aswell as nucleic acid molecules that encode such homologs.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode identical or similar (conserved) amino acidsequences, due to the degeneracy of the genetic code. Changes in anucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein. Such homologous peptides can, for example, possess at least75%, 80%, 90%, 95%, 98%, or 99% sequence identity determined by thismethod. When less than the entire sequence is being compared forsequence identity, homologs can, for example, possess at least 75%, 85%90%, 95%, 98% or 99% sequence identity over short windows of 10-20 aminoacids. Methods for determining sequence identity over such short windowscan be found at the NCBI web site. One of skill in the art willappreciate that these sequence identity ranges are provided for guidanceonly; it is possible that significant homologs or other variants can beobtained that fall outside the ranges provided.

Subject: Living multicellular vertebrate organisms, a category whichincludes, both human and veterinary subjects that require an increase inthe desired biological effect. Examples include, but are not limited to:humans, apes, dogs, cats, mice, rats, rabbits, horses, pigs, and cows.

Therapeutically Effective Amount: An amount sufficient to achieve adesired biological effect, for example an amount that is effective todecrease the size (i.e. volume), side effects and/or metastasis ofprostate cancer. In one example, it is an amount sufficient to decreasethe symptoms or effects of a prostate carcinoma, such as the size of thetumor. In particular examples, it is an amount effective to decrease thesize of a prostate tumor and/or prostate metastasis by at least 30%,40%, 50%, 70%, 80%, 90%, 95%, 99% or even 100% (complete elimination ofthe tumor).

In particular examples, it is an amount of a modified PA moleculeeffective to decrease a prostate tumor and/or an amount of prostatecancer cells lysed by a modified PA, such as in a subject to whom it isadministered, for example a subject having one or more prostatecarcinomas. In other examples, it is an amount of a modified PA moleculeand/or an amount of prostate cancer cells lysed by such a modified PAmolecule, effective to decrease the metastasis of a prostate carcinoma.

In one embodiment, the therapeutically effective amount also includes aquantity of modified PA and/or an amount of prostate cancer cells lysedby a modified PA sufficient to achieve a desired effect in a subjectbeing treated. For instance, these can be an amount necessary to improvesigns and/or symptoms a disease such as cancer, for example prostatecancer.

An effective amount of modified PA and/or prostate cancer cells lysed bysuch a modified PA molecule can be administered in a single dose, or inseveral doses, for example daily, during a course of treatment. However,the effective amount of will be dependent on the subject being treated,the severity and type of the condition being treated, and the manner ofadministration. For example, a therapeutically effective amount ofmodified PA can vary from about 1-10 mg per 70 kg body weight, forexample about 2.8 mg, if administered iv and about 10-100 mg per 70 kgbody weight, for example about 28 mg, if administered intraprostaticallyor intratumorally. In addition, a therapeutically effective amount ofprostate cancer cells lysed by PA (variant or wild-type) can vary fromabout 10⁶ to 10⁸ cells.

Therapeutically effective dose: In one example, a dose of modified PAsufficient to decrease tumor cell volume, such as a prostate carcinoma,in a subject to whom it is administered, resulting in a regression of apathological condition, or which is capable of relieving signs orsymptoms caused by the condition. In a particular example, it is a doseof modified PA sufficient to decrease metastasis of a prostate cancer.

In yet another example, it is a dose of cell lysate resulting fromcontact of cells with a modified PA sufficient to decrease tumor cellvolume, such as a prostate carcinoma, in a subject to whom it isadministered, resulting in a regression of a pathological condition, orwhich is capable of relieving signs or symptoms caused by the condition.In a particular example, it is a dose of cell lysate resulting fromcontact of cells with a modified or wild-type PA sufficient to decreasemetastasis of a prostate cancer.

Tumor: A neoplasm. Includes solid and hematological (or liquid) tumors.

Examples of hematological tumors include, but are not limited to:leukemias, including acute leukemias (such as acute lymphocyticleukemia, acute myelocytic leukemia, acute myelogenous leukemia andmyeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (including low-, intermediate-, and high-grade),multiple myeloma, Waldenstrdm's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, mantle cell lymphoma and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include, butare not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, andCNS tumors (such as a glioma, astrocytoma, medulloblastoma,craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Transgenic Cell: Transformed cells which contain foreign, non-nativeDNA.

Transgenic mammal: Transformed mammals which contain foreign, non-nativeDNA. In one embodiment, the non-native DNA is a modified PA whichincludes a PSA cleavage site, such as SEQ ID NO: 3, or a nucleic acidsequence which encodes for a protein shown in SEQ ID NOS: 24 or 25.

Variants or fragments or fusion proteins: The production of modified PAprotein can be accomplished in a variety of ways (for example seeExamples 12 and 16). DNA sequences which encode for a modified PAprotein or fusion protein, or a fragment or variant of a protein (forexample a fragment or variant having 80%, 90% or 95% sequence identityto a modified PA) can be engineered to allow the protein to be expressedin eukaryotic cells or organisms, bacteria, insects, and/or plants. Toobtain expression, the DNA sequence can be altered and operably linkedto other regulatory sequences. The final product, which contains theregulatory sequences and the therapeutic modified PA protein, isreferred to as a vector. This vector can be introduced into eukaryotic,bacteria, insect, and/or plant cells. Once inside the cell the vectorallows the protein to be produced.

A fusion protein which includes a modified PA, (or variants,polymorphisms, mutants, or fragments thereof) linked to other amino acidsequences that do not inhibit the desired activity of the protein, forexample the ability to lyse PSA-secreting cells. In one example, theother amino acid sequences are no more than 5, 6, 7, 8, 9, 10, 20, 30,or 50 amino acid residues in length.

One of ordinary skill in the art will appreciate that the DNA can bealtered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes a variant PA toxin. Such variants canbe variants optimized for codon preference in a host cell used toexpress the protein, or other sequence changes that facilitateexpression.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector can also include one or more selectablemarker genes and other genetic elements known in the art.

Additional definitions of terms commonly used in molecular genetics canbe found in Benjamin Lewin, Genes V published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Variant Proaerolysin Molecules

Bacterial toxins, such as aerolysin produced by Aeromonas hydrophiliaand α-hemolysin produced by Staph aureus, are beta-sheet proteins thatoligomerize in the plasma membrane to produce pores that lead to rapidcytolytic cell death (FIG. 1). Pore formation physically disrupts thecell membranes, and results in death of cells in all phases of the cellcycle, including non-proliferating cells (i.e. GO arrested). However,wild-type aerolysin kills cells indiscriminately. Herein disclosed is aninactive protoxin form of aerolysin (a variant PA) that can be targetedto, and activated by, prostate cancer specific proteins. One advantageof the disclosed variant PA molecules for treatment of localized andmetastatic prostate cancer, is that it combines a proliferationindependent therapy with prostate-specific drug delivery, resulting inminimal side effects to patients. One skilled in the art will understandthat other protoxins, such as Clostridium septicum alpha toxin, Bacillusthuringiensis delta-toxin, and human perforin, can be substituted forproaerolysin.

Disclosed herein are variant PA molecules, including both DNA andprotein sequences, which include a prostate-specific protease cleavagesequence. Examples of prostate-specific protease cleavage sequencesinclude, but are not limited to: PSA, PSMA, and HK2 cleavage sequences.The prostate-specific protease cleavage sequence functionally replacesthe native furin cleavage site of PA (for example, see FIGS. 5B-I). Thisreplacement results in a proaerolysin variant that only becomescytolytically active in the presence of enzymatically active protease,such as PSA, PSMA, or hK2. PSA is a serine protease with the ability torecognize and hydrolyze specific peptide sequences. It is secreted bynormal and malignant prostate cells in an enzymatically active form andbecomes inactivated upon entering the circulation. Since neither bloodnor normal tissue other than the prostate contains enzymatically activePSA, the proteolytic activity of PSA was used to activate protoxins atsites of prostate cancer. Any PSA, PSMA, or hK2 cleavage site can beused. Examples of PSA cleavage sites include, but are not limited to,those shown in SEQ ID NOS: 5, 8, 11, and 14-21. In a particular example,the PSA cleavage site includes SEQ ID NO: 5.

In some examples, the furin cleavage site of PA (amino acids 427-432 ofSEQ ID NO: 2) is deleted and a prostate-specific protease cleavage site,such as a PSA cleavage site, is inserted (for example, see FIG. 5B). Inother examples, the furin cleavage site of PA is mutated and aprostate-specific protease cleavage site, such as a PSA cleavage site,inserted with in, or added to the N- or C-terminus of the furin site(for example, see FIG. 5C).

Also disclosed are variant PA molecules in which the PA binding domainis functionally deleted (for example, see FIGS. 5D-M). Such variant PAmolecules can contain a native furin cleavage site (for example, seeFIGS. 5J-M), whereby targeting to prostate cells is achieved byfunctionally replacing the PA binding domain with a prostate-tissuespecific binding domain. Alternatively, variant PA molecules contain aprostate-specific protease cleavage site (for example, see FIGS. 5D-I),whereby activation of the protoxin primarily occurs in cells thatsecrete a prostate-specific protease. The PA binding domain includesabout amino acids 1-83 of SEQ ID NO: 2. The binding domain can befunctionally deleted using any method known in the art, for example bydeletion of all or some of the amino acids of the binding domain, suchas deletion of amino acids 1-83 of SEQ ID NO: 2 or 4 (for example seeFIG. 5G), or such as deletion of one or more amino acids shown as aminoacids 45-66 of SEQ ID NO: 2 or 4 (for example see FIG. 5D where the *represents one or more deletions). In other examples, the binding domainis functionally deleted by introduction of one or more site-specificmutations into the variant PA sequence, such as W45A, 147E, M57A, Y61A,and K66Q of SEQ ID NO: 2 or 4 (for example see FIG. 5D, where the *represents one or more mutations).

Variant PA molecules which include a prostate-tissue specific bindingdomain which functionally substitutes for the native PA binding domainare disclosed (for example, see FIGS. 5E, 5F, 5H and 5I-M). The use ofone or more prostate-tissue specific binding domains can increasetargeting of the disclosed variant PA molecules to the prostate cellsand its metastases. Several prostate-tissue specific binding domains areknown. Examples include, but are not limited to a luteinizing hormonereleasing hormone (LHRH) sequence, such as those shown in SEQ ID NOS: 22and 23, and antibodies that recognize PSA and/or PSMA.

One or more prostate-tissue specific binding domains can be linked toone or more amino acids of the disclosed variant PA molecules, butideally, do not interfere significantly with the ability of the variantPA to be activated by a prostate-specific protease such as PSA, and theability to form pores in cell membranes. For example, prostate tissuespecific binding domains can be linked or inserted at an N- and/orC-terminus of a variant PA (for example, see FIGS. 5E and 5H). In someexamples, the native binding domain of PA is deleted (i.e. amino acids1-83 of SEQ ID NO: 2 or 4), such that attachment or linking of aprostate tissue specific binding domain to the N-terminus results inattachment to amino acid 84 of SEQ ID NO: 2 or 4 (for example, see FIGS.5H and 5L). In other examples, smaller deletions or point mutations areintroduced into the native binding domain of PA, such that attachment orlinking of a prostate tissue specific binding domain to the N-terminusresults in attachment to amino acid 1 of SEQ ID NO: 2 or 4 (or whicheveramino acid is N terminal following functional deletion of the native PAbinding domain) (for example, see FIGS. 5E and 5I). In some examples,the N-terminal amino acid of PA is changed to a Cys or other amino acidto before attaching a prostate-tissue specific binding domain, to assistin linking the prostate-tissue specific binding domain to the variant PAprotein.

Alternatively or in addition, one or more prostate tissue specificbinding domains can be attached or linked to other amino acids of avariant PA molecule, such as amino acid 215 or 300 of SEQ ID NO: 2 or 4(for example, see FIGS. 5F, 5I, 5K and 5M). In some examples, a Cysamino acid replaces the native amino acid at that position. For example,the following changes can be made to SEQ ID NO: 2 or 4: Tyr215Cys orAla300Cys. In one example, where the prostate tissue specific bindingdomain is an antibody, crosslinking can be used to attach antibodies toa variant PA, for example by reacting amino groups on the antibody withcysteine located in the PA variant (such as amino acids Cys19, Cys75,Cys159, and/or Cys164 of SEQ ID NO: 2).

Also disclosed are particular variant PA molecules, such as those shownin SEQ ID NOS: 3, 4, 6, 7, 9, 10, 12, 13, 24 and 25.

In some examples the disclosed variant PA molecules are linked orimmobilized to a surface, such as a bead. The bead can also include aprostate-specific ligand to enhance targeting to a prostate cell, suchas a localized or metastasized prostate cancer cell.

Treatment of Prostate Cancer Using Modified Proaerolysin

The variant PA molecules disclosed and discussed above are specificallyactivated to potent cytotoxins within prostate cancer sites via theproteolytic activity of prostate-specific proteases such as PSA, PSMA,and hK2. Targeting in some examples is achieved by including one or moreprostate-tissue specific binding domains, such as LHRH peptide which canbind to its cognate LHRH receptor expressed by prostate cancer cells, orPSMA or LHRH antibodies, which can bind to PSMA or LHRH expressed on thesurface of prostate cancer cells. One skilled in the art will recognizethat the use of a variant PA molecule which includes a furin cleavagesite and an LHRH peptide or antibody, can be used to treat other cancerswhich express LHRH receptors, such as melanoma and cancers of thebreast, ovary and lung, using the variant PA molecules and methodsdisclosed herein. Furthermore, one skilled in the art will recognizethat the use of a variant PA molecule which includes a furin or PSMAcleavage site, and/or a PSMA antibody, can be used to treat othercancers in which PSMA is expressed (e.g. in the vasculature of thetumor), such as cancers of the breast, colon, kidney, bladder and brain,using the variant PA molecules and methods disclosed herein.

The disclosed variant PA molecules, such as nucleic acids and/orproteins, can be administered locally or systemically using any methodknown in the art, to subjects having localized or metastatic prostatecancer. In addition, the disclosed variant PA molecules can beadministered to a subject for immunostimulatory therapy. Due to thespecificity of binding and activation of the disclosed variant PAmolecules, local and systemic administration should have minimal effecton a patient's normal tissues and ideally produce little to no sideeffects.

In one example, the disclosed variant PA molecules are injected into theprostate gland (intraprostatically) and/or into the prostate tumor(intratumorally) in a subject having prostate cancer, such as alocalized tumor. Such localized injection and subsequent lysis ofprostate cancer cells within the prostate gland can produce animmunostimulatory effect leading to a decrease or elimination ofmicrometastatic disease in treated subjects. In this way, systemicdisease is treated or reduced through a minimally toxic, locally appliedtherapy.

In addition, or alternatively, the disclosed variant PA molecules can beadministered systemically, for example intravenously, intramuscularly,subcutaneously, or orally, to a subject having prostate cancer, such asa metastatic prostate tumor. Systemic therapy can also have animmunostimulatory anti-tumor effect. The disclosed variant PA moleculeswhich include a PSA-cleavage site are not hydrolyzed by serum proteasesor enzymatically inactive PSA within the blood. Instead, theunhydrolyzed disclosed variant PA molecules are delivered via the bloodto the extracellular fluid within metastatic cancer deposits where theycan be hydrolyzed to the active therapeutic toxin by the enzymaticallyactive PSA secreted by these prostate cancer cells. Once hydrolyzed, theliberated toxin enters PSA-producing and non-producing bystander cellsin the immediate vicinity due to its high membrane penetrating abilityand induces the cytolytic death of these cells.

An additional method for systemically treating prostate cancer in asubject is also disclosed. In this method, prostate cancer cells areremoved from the subject having prostate cancer, such as a metastaticprostate tumor. Alternatively or in addition, established prostatecancer cell lines can be used. Examples of prostate cancer cell linesthat can be used include, but are not limited to: PSA-producing cellssuch as LNCaP (such as ATTC Nos. CRL-1740 and CRL-10995) and CWR22R(ATCC No. CRL-2505 and Nagabhushan et al., Cancer Res. 56(13):3042-6,1996), or PSA non-producing cells such as PC-3 (ATCC No. CRL-1435) andDU 145 (ATCC No. HTB-81). The removed cells or cell lines are incubatedor contacted with the disclosed variant PA molecules. This incubationresults in lysis of the cells by the variant PA molecules, andproduction of a cell lysate which is administered to the subject. In oneexample, the method further includes administration of immunostimulatoryfactors, lysates from prostate cancer cells engineered to produceimmunostimulatory factors, and/or irradiated prostate cancer cells(including prostate cancer cells engineered to produce immunostimulatoryfactors). Examples of immunostimulatory factors include, but are notlimited to: granulocyte macrophage colony stimulatory factor (GM-CSF);members of the interleukin family of proteins such as but not limited tointerleukin-2 and interleukin-6, granulocyte colony stimulatory factor(G-CSF); and members of interferon family such as interferon alpha, betaor gamma. Administration of such materials to a subject can besimultaneous with the cell lysate (co-administration), beforeadministration of the cell lysate, and/or subsequent to administrationof the cell lysate.

In one example, such administration enhances the ability of a subject todecrease the volume of a prostate tumor and/or a metastatic tumor. Forexample, the disclosed methods can reduce prostate tumor cell volumeand/or a metastatic tumor cell volume, such as by at least 10%, forexample by at least 20% or more. In addition, the disclosed methods canresult in a decrease in the symptoms associated with a prostate tumorand/or a metastatic prostate tumor.

The disclosed variant PA molecules can be administered as a singlemodality therapy or used in combination with other therapies, such asradiation therapy and/or androgen ablative therapies (such as LHRHreceptor agonists/antagonists, antiandrogens, estrogens, adrenal steroidsynthesis inhibitors ketoconazole and aminoglutethimide). In addition,administration of the disclosed variant PA molecules can be alone, or incombination with a pharmaceutically acceptable carrier, and/or incombination with other therapeutic compounds, such as those that reducethe production of antibodies to the administered variant PA proteins(for example Rituximab and steroids) and other anti-tumor agents.

Disclosure of certain specific examples is not meant to exclude otherembodiments. In addition, any treatments described herein are notnecessarily exclusive of other treatment, but can be combined with otherbioactive agents or treatment modalities.

Example 1 Generation of PSA-Activated Proaerolysin Toxins

This example describes methods used to produce the variant proaerolysintoxins shown in Table 1, which are activated by PSA. One skilled in theart will understand that similar methods can be used to produce othervariant proaerolysin proteins which are activated by PSA or any otherprostate-specific protease. Such proteins can be produced bysubstituting the furin sequence of proaerolysin with a prostate-specificprotease cleavage site, such as a PSA-specific cleavage sequence (seeExample 9). TABLE 1 PSA-specific proaerolysin variants Comparison to wtProaerolysin Mutant Change(s) made ADSKVRRARSVDGAGQGLRLEIPLD (SEQ ID NO)(SEQ ID NO) (aa 424-448 of SEQ ID NO:2) PSA-PA1 KVRRARADSHSSKLQSVDGAGQGLRLEIPLD (3 & 4) (aa 427-432 of SEQ ID NO:2) (aa424-448 of SEQ ID NO:4) changed to HSSKLQ (5) PSA-1K KVRRARSVADSHSSKLQSADGAGQGRLEIPLD (6 & 7) (aa 427-434 of SEQ ID NO:2) (aa 424-448of SEQ ID NO:7) changed to HSSKLQSA (8) PSA-PA2 KVRRARADSQFYSSNSVDGAGQGLRLEIPLD (9 & 10) (aa 427-432 of SEQ ID NO:2) (aa424-448 of SEQ ID NO:10) changed to QFYSSN (11) PSA-PA3 KVRRARADSGISSFQSSVDGAGGLRLEIPLD (12 & 13) (aa 427-432 of SEQ ID NO:2) (aa424-448 of SEQ ID NO:13) changed to GISSFQS (14)

The variant or modified proaerolysins (PA) shown in Table 1, include aproaerolysin sequence (wild-type PA shown in SEQ ID NOS: 1 and 2) inwhich the six amino acid furin protease recognition site (amino acids427-432 of SEQ ID NO: 2) was replaced with a PSA substrate. For example,the variant proaerolysin (PA) toxin termed PSA-PA1 (SEQ ID NOS: 3 and4), includes a PA sequence in which the furin cleavage site was replacedby the PSA substrate HSSKLQ (SEQ ID NO: 5).

Recombinant PCR was used to substitute the furin site of aerolysin(amino acids 427-432 of SEQ ID NO: 2) with a PSA-specific cleavage site(SEQ ID NO: 5, 8, 11 or 14) using methods previously described (Valletteet al., Nucl. Acids Res. 17:723-33, 1988). Briefly, recombinant PCR wasperformed in a final volume of 50 μl which contained 0.2 mMdeoxynucleoside triphosphate (dNTPs), 0.5 μM forward and reverseprimers, 0.1 μg template DNA and 2.5 units cloned pfu polymerase in pfuReaction Buffer [20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2mM MgSO₄, 0.1% Triton X-100, and 0.1 mg/ml BSA].

Screening transformed cells for the proaerolysin insert was performed byPCR using Taq polymerase. A cocktail was prepared in PCR reaction buffer[50 mM KCl, 1.5 mM MgCl₂, and 10 mM Tris-HCl (pH 9.0)] containing 0.2 mMdNTPs, 0.5 μM forward and reverse primers and 5 units of Taq polymerase.Ten μl samples of this cocktail were aliquoted into 0.2 ml tubes andtransformed cells were added using sterile toothpicks.

The final PCR products were digested using appropriate restrictionenzymes, then ligated into the cloning vector pTZ18u (BioRad) foramplification. Briefly, restriction digests were performed at 37° C. for90 minutes in Pharmacia One-Phor-All buffer [10 mM Tris-acetate (pH7.5), 10 mM Mg-acetate, and 50 mM K-acetate] containing about one unitof restriction enzyme for every μg of DNA. The resulting insert, andpTZ18u vector DNA, were mixed together in a ratio of approximately 5:1and heated at 45° C. for 15 minutes. Subsequently, the samples werediluted in One-Phor All buffer and ATP added to a final concentration of1 mM for cohesive-end ligations or 0.5 mM for blunt-end ligations. Then,11 units of T4 DNA ligase were added to each sample and the samplesmixed gently. Ligations were carried out at 13° C. for 4 hours(cohesive-end ligations) or 16 hours (blunt-end ligations).

DNA sequencing was performed to ensure the correct substitutions weremade. The insert was subsequently isolated from the cloning vector andsubcloned into the broad-host-range plasmid pMMB66HE (Furste et al.,Gene 48:119-131, 1986) for expression in E. coli. E. coli DH5α cellswere made competent using the CaCl₂ wash method described previously(Cohen et al. Proc. Natl. Acad. Sci. USA 69:2110-4, 1972). Cells inlog-phase (OD₆₀₀=0.4-0.7) were harvested by centrifugation and washed in¼ volume of cold 100 mM MgCl₂. The cells were pelleted again, andresuspended in two volumes of cold 100 mM CaCl₂. The cells were thenincubated on ice for approximately 45 minutes. The cells were thencentrifuged and resuspended in 1/10 volume of 100 mM CaCl₂. Incubationcontinued for an additional 45 minutes before the addition of glycerolto a final concentration of 15%. Competent cells were stored at −70° C.until use.

Transformation of recombinant plasmids into competent E. coli cells wasperformed according to the method of Inoue et al. (Gene 96: 23-8, 1990).Competent cells (200 μl aliquots) were incubated with 0.5-10 ng of DNAfor one hour on ice. The cells were then subjected to heat shock at 42°C. for 4 minutes. The cells were quickly transferred back onto ice for 5minutes. Subsequently, 500 μl of LB media was added to each sample andthe cells incubated for 1 hour at 37° C. with mild agitation. Aliquots(150 μl) were plated onto LB agar containing 50 μg/ml ampicillin. Theseplates were incubated ON at 37° C.

Recombinant pMMB66HE clones were transferred into Aeromonas salmonicidastrain CB3 (see Buckley, Biochem. Cell. Biol. 68:221-4, 1990) byconjugation using the filter-mating technique of Harayama et al. (Mol.Gen. Genet. 180:47-56, 1980). Use of this protease-deficient strain ofA. salmonicida resulted in production of proaerolysin variants that werenot contaminated by activated aerolysin and resulted in production oflarge quantities of protein. The final proaerolysin protein was purifiedby hydroxyapatite chromatography and ion exchange chromatography aspreviously described (Buckley, Biochem. Cell. Biol. 68:221-4, 1990).This method resulted in preparations of the proaerolysin proteinsidentical from batch to batch.

Example 2 PSA-PA1 Specifically Lyses PSA-Producing Cells In Vitro

This example describes methods used to determine the specificity of theproaerolysin variants described in Example 1. Such methods can be usedto test the specificity of any proaerolysin variant that includes aPSA-specific cleavage site.

PSA-PA1 toxin was tested against PSA-producing LNCaP cells (AmericanType Culture Collection, Manassas, Va.) and non-PSA-producing TSU cells(Dr. T. Itzumi, Teikyo University, Japan). Cells were incubated in thepresence of 10⁻¹² M to 10⁻⁶ M toxin for 24 hours. Subsequently, cellswere counted and scored for percent viable cells based on ability toexclude Trypan Blue. Concentration required to kill 50% of cells (IC₅₀)was determined for the toxin against both LNCaP and TSU lines.

The LD₅₀ for PSA-PA1 against PSA-producing cells was 10⁻¹⁰ M. Incontrast, against non-PSA producing TSU cells the LD₅₀ was ˜5×10⁻⁸ M.This result demonstrates that the PSA-PA1 toxin is specificallyactivated by PSA as evidenced by a 500-fold difference in toxicityagainst PSA-producing versus non-PSA producing human cancer cell lines.

Example 3 PSA-PA1 is Not Activated in Blood Containing PSA

The disclosed prostate-specific protease-activated variant proaerolysinpeptides, such as those described in Example 1, can be injectedintraprostatically (or intratumorally) as local therapy for prostatecancer. The toxin can also be injected intravenously (orintramuscularly) as systemic therapy for metastatic prostate cancer.Those variant PA molecules which include a PSA site should not beactivated in blood, because PSA is enzymatically inactivated in theblood due to the presence of a large molar excess of serum proteaseinhibitors such as α₁-antichymotrypsin and α₂-macroglobulin.

To test for non-specific activation of PSA-PA1 by other serum proteasesand PSA in human serum, a sensitive hemolysis assay was performed asfollows. Red blood cells (RBCs, 2% v/v) were added to plasma or buffercontaining PSA-PAL toxin±PSA. The extent of hemolysis was assayed bymeasuring release of hemoglobin into the supernatant. Addition of 0.1%Triton results in 100% hemolysis within a few seconds and was used asthe positive control. Amount of hydrolysis was expressed as a ratio ofsample absorbance at 540 nm to absorbance of Triton treated sample.Pre-incubation of the PSA-PA1 toxin (10⁻⁸ M) with PSA in aqueous bufferalone for 1 hour prior to adding RBCs resulted in ˜45% hemolysis (FIG.2).

To determine whether PSA-PA1 becomes activated in human plasma, PSA-PA1toxin (10-⁸ M) was incubated in 50% human plasma for 1 hour. In arelated experiment, excess PSA (10,000 ng/ml) was first added to thehuman plasma and allowed to incubate for several hours. The PSA-PA1containing plasma±PSA was then incubated with human RBCs (2% v/v). Theaddition of PSA-PA1 to human plasma, or human plasma spiked with highconcentration of PSA, resulted in no appreciable hemolysis (i.e. <1% ofTriton control) (FIG. 2). These results demonstrate that PSA-PA1 can beadministered systemically without any significant activation in theblood, even if the blood contains measurable PSA.

Example 4 In Vitro and In Vivo Toxicity Proaerolysin Variants

This example describes methods used to determine the in vitro and invivo toxicity of the disclosed modified proaerolysin proteins. Suchmethods can be used to measure the toxicity of any prostate-specificprotease-cleavable proaerolysin variant protein.

To determine in vitro toxicity, a cell viability assay was performed asfollows. El4 mouse T-cell lymphoma cells (ATCC TIB-39) were cultured at10⁺⁵ cells per well in MTS/PMS Cell Titer 96 (Promega). Proaerolysinvariants at 1×10⁻¹³ M-1×10⁻⁷ M was added as shown in FIG. 3, andincubated with the cells for 4 hours at 37° C. Cell viability wassubsequently determined by reading the plate on a plate reader, asdirected by the manufacturer of the MTS/PMS kit. As shown in FIG. 3, theproaerolysin variants are less toxic than wild-type proaerolysin, withan LC₅₀ of 4×10⁻⁹ (PSA-PA1), 1×10⁻⁹ (PSA-1K), and 1×10⁻⁷ (PSA-PA2), incontrast to an LC₅₀ of 1.5×10⁻¹⁰ for wild-type.

To determine in vivo toxicity, proaerolysin variants were administeredto mice intravenously. Wild-type proaerolysin (SEQ ID NO: 2) was highlytoxic to mice; a dose of 1 μg caused death within one hour and the LD₁₀₀at 24 hours (i.e. the dose that kills 100% of animals within 24 hours)following a single IV injection was 0.1 μg. In contrast, the LD₁₀₀ ofPSA-PA1 (SEQ ID NO: 4) at 24 hours post injection was 25-fold higher(i.e. 2.5 μg total dose).

Example 5

PSA-PA1 Reduces PSA-Secreting Tumor Cell Volume

On the basis of the toxicity data described in Example 4, a series ofLNCaP bearing mice (human LNCaP prostate cancer xenografts which producePSA) and a series of SN12C bearing mice (control mice which have a humanrenal carcinoma xenograft which does not produce PSA) were administereda single 100 μl intratumoral injection of 0.25-25 μg PSA-PA1 (0.1-10times the LD₁₀₀ dose). At 48 hours post injection, tumors wereharvested, fixed and stained with H&E, and for Ki-67 (proliferativeindex) and Tunel (apoptotic index). The percent of tumor within samplewas determined by calculating the ratio of viable tumor to total tumorarea following image analysis of thin tumor sections.

As shown in FIG. 4, administration of 2.5-25 μg of PSA-PA1, reducedtumor cell volume by 85-99%, while <20% reduction was observed at 0.25μg of PSA-PA1. In contrast, when mice bearing non-PSA producing SN12Chuman renal carcinoma xenografts (cells provided by Dr. Isaiah FidlerAnderson Cancer Center, Houston Tex.), were injected intratumorally withPSA-PA1, no significant reduction (i.e. <25%) in percent of viable tumorwas observed over the same dose range (FIG. 4).

In control tumors (SN12C mice), the proliferative index was ˜40% 48hours post administration of intratumoral PSA-PA1. In contrast, in theresponding PSA-PA1 treated tumors the percent of Ki-67 positive cellswas <1% at 48 hours. In addition, in the control tumors the apoptoticindex was <1% 48 hours post administration of intratumoral PSA-PA1,while in the PSA-PA1 treated tumors all cells were positive by 48 hours(i.e. apoptotic index>99%).

These results demonstrate that the PSA-PA1 toxin efficiently and rapidlykills PSA-producing cells and this cytotoxicity in vivo is specific anddependent on the presence of PSA present in the extracellular fluid oftumors.

Example 6 Targeting the Protoxin Via Prostate-Specific Binding Domains

This example describes methods to generate and use variant prostatespecific protease-activated proaerolysin toxins in which the wild-type(or native) PA binding domain (approximately amino acids 1-83 of SEQ IDNO: 2) is functionally replaced with a prostate-tissue specific bindingdomain, such as the LHRH peptide. The native binding domain of PA can befunctionally deleted by any method known in the art, for example bydeletion of about amino acids 1-83 of SEQ ID NO: 2 (or fragmentsthereof, such as 45-66), or by insertion of mutations which decrease theability of PA to concentrate in cell membranes.

The N-terminal GPI-anchor protein binding domain of PA (approximatelyamino acids 1-83 of SEQ ID NO: 2) can be functionally deleted (forexample by deletion of amino acids 1-83 or by insertion of mutationswhich render the domain non-functional) without significantly effectingpore-formation. A binding domain is needed, however, to concentrate thetoxin in the cell membrane. Mutant proteins that lack the binding domainare cytolytic, but only when cells are exposed to several orders ofmagnitude higher concentrations of toxin. Most in vivo toxicity of wildtype and variant PSA-activated PA described above in Example 4 is due tonon-specific binding to GPI-anchor proteins expressed by most mammaliancells. To generate a more specific, and less systemically toxicprotoxin, the non-specific GPI-anchor protein binding domain ofproaerolysin can be functionally deleted and replaced with a prostatetissue-specific binding domain.

Antibodies

An example of a binding moiety that can be used to target a modifiedproaerolysin protein with high specificity to prostate tissue is afusion protein which includes a single chain antibody to aprostate-specific membrane protein, and antibodies that recognize PSMAor LHRH fused to the toxin domains. Ideally, attachment of the antibodyto a modified PA molecule will not significantly interfere with theability of the molecule to bind to and concentrate in a cell membrane,resulting in cell death. Antibodies can be attached to the N- orC-terminus of a modified PA using gene fusion methods well known in theart (for example see Debinski and Pastan, Clin. Cancer Res. 1: 1015-22,1995). For example, the antibody could replace the native small lobe ofproaerolysin (amino acids 1-83 of SEQ ID NO: 2), or the antibody couldbe added to a PA molecule having mutations in the native binding domain.Such a modified proaerolysin can also include a PSA activation sequenceto increase specificity. In one example, the antibody is a single chainantibody to PSMA fused to the toxin domain of PA.

Alternatively, antibodies or FAB fragments can be attached to a modifiedPA by covalent crosslinking (for example see Woo et al., Arch. Pharm.Res. 22(5):459-63, 1999 and Debinski and Pastan, Clin. Cancer Res.1(9):1015-22, 1995). Crosslinking can be non-specific, for example byusing a homobifunctional-lysine-reactive crosslinking agent, or it canbe specific, for example by using a crosslinking agent that reacts withamino groups on the antibody and with cysteine located in theproaerolysin variant (such as amino acids Cys 19, Cys75, Cys159, and/orCys164 of SEQ ID NO: 2).

Ligands

Other binding moieties that can be used are small peptide ligands thatbind to their cognate receptor expressed on the membrane of prostatecancer cells. An example includes, but is not limited to, natural andsynthetic luteinizing hormone releasing hormone (LHRH) agonist peptides(for example see Genbank Accession No. CAA25526 and SEQ ID NOS: 22 and23), which bind with high affinity to LHRH receptors, and peptides thatcan bind selectively to PMSA. LHRH receptors are expressed by a highpercentage of human prostate cancers, but not by hematopoeitic cells.This differential expression provides binding specificity.

It is known that certain residues of LHRH, such as the Gly at the 6thposition (Gly6), can be substituted without compromising receptorbinding affinity (Janaky et al., Proc. Natl. Acad. Sci. USA 89:972-6,1992; Nechushtan et al., J. Biol. Chem., 272:11597-603, 1997).Therefore, a variant PA toxin (in which the native binding domain isfunctionally deleted) can be produced which is covalently coupled topurified LHRH D-Lys6 (at the epsilon amine of this lysine).

LHRH D-Lys6 (SEQ ID NO: 23) can be attached to various portions of amodified proaerolysin protein having a functionally deleted bindingdomain. Ideally however, such placement will not significantly interferewith the ability of the toxin to insert into the membrane to form apore. For example, the epsilon amine of the D-Lys6 analog can be coupledto the amino terminus of the modified proaerolysin via a dicarboxylicacid linker. Cleavage by furin or a prostate-specific protease(depending on which cleavage site is present in the proaerolysinpeptide) will result in release of the C-terminal inhibitory portionwhile the toxin remains bound to the LHRH receptor.

Alternatively or in addition, the epsilon amine of the D-Lys6 analog ofLHRH can be coupled directly to the C-terminal carboxyl of the modifiedproaerolysin lacking a functional wild type binding domain, by theaddition of a Cys to the C-terminus of the modified PA, thencrosslinking this Cys to the epsilon amine of the D-Lys6 analog of LHRH.This coupling will produce a derivative PA protein in which the LHRHpeptide is attached to the C-terminal inhibitory domain of PA. Cleavageby furin or by a prostate specific protease, such as PSA, will liberatethe toxin and leave the inhibitory fragment bound to the LHRH receptor.Therefore, pore formation should not be inhibited by tight binding tothe receptor. In addition, recombinant fusion proteins can be producedin which modified LHRH peptides are fused to both the N- and C-terminusof the modified PA toxin lacking a functional native binding domain.

In other examples, a cysteine residue is introduced into the 6thposition of the LHRH peptide and the peptide attached to the modified PAtoxins via a disulfide bridge, for example at amino acids 215 and/or 300of SEQ ID NO: 2, wherein amino acids 215 and/or 300 has been mutated toa cysteine. In another example, a recombinant protein is produced inwhich LHRH peptide is fused to the amino terminus of the modified PAtoxin.

The resulting modified proaerolysin proteins which include a prostatetissue specific binding domain functionally substituted for the nativePA binding domain, are tested in vitro and in vivo for bindingspecificity and toxin activation, using the methods described inExamples 1-5.

To demonstrate that prostate-tissue specific binding domains, such asLHRH or PSMA, can be functionally substituted for the native PA bindingdomain the following experiments can be performed. The methods describedbelow describe the use of a molecule in which the native PA bindingdomain has been deleted, and LHRH linked to the resulting proaerolysin.However, similar methods can be used to test any variant PA molecule,such as molecules in which other prostate-tissue specific bindingdomains are used, and where the PA binding domain is mutated (forexample by insertion of one or more of the following mutations: W45A,147E, M57A, Y61A, K66Q, and W324A) instead of deleted.

LHRH-proaerolysin proteins containing the native PA furin activationsequence are produced. The specificity of binding of these toxins toLHRH receptor positive (LNCaP) and negative (TSU) cells is comparedusing methods described in Example 2. Both cell lines activate the wildtype, furin-activation-site-containing PA. Therefore, while each linemay activate the LHRH-proaerolysin proteins, the ideal peptide is onethat is toxic at low concentrations to LHRH receptor positive cells.Using these methods, regions of proaerolysin to which the LHRH peptidecan be attached without interfering with channel formation by the toxinare identified.

To demonstrate that LHRH-proaerolysin proteins both bind to LHRHreceptor and are activated by PSA, toxins that contain a PSA-activationsite instead of the furin site are generated using the methods describedin Example 1. The activation of these toxins by LHRH positive,PSA-producing LNCaP cells is compared to activation by LHRH receptornegative, PSA non-producing TSU cells, using the methods described inExample 2.

LHRH-PSA-activated proaerolysin toxins are tested in vivo to demonstratethat introduction of the LHRH binding moiety results in decreasednon-specific toxicity and increased targeting ability. As describedabove, the LD₁₀₀ of these toxins following a single intravenousinjection is determined and compared to the corresponding non-LHRHcontaining, PSA-activated toxin, using the methods described in Example4. Subsequently, the toxin is administered at varying doses, such as 0.1μg to 1 mg, intravenously to LNCaP bearing animals to demonstrate thatan enhanced anti-tumor effect is observed at higher, less toxic doses oftoxin.

Example 7 Determination of Proaerolysin Antigenicity

This example provides methods to determine if the modified proaerolysinpeptides disclosed herein are antigenic. In addition, methods to reducepotential antigenicity are disclosed.

As described in the Examples above, intratumoral injection of PSA-PA1(SEQ ID NO: 4) demonstrates the usefulness of the toxin as therapy forlocalized prostate cancer via intraprostatic injection. However, such atherapy can also be administered by other routes, such as intravenous(iv), intramuscularly, orally, etc., as a systemic therapy formetastatic prostate cancer. However, systemic administration of thevariant PA peptides disclosed herein may result in the development of aneutralizing antibody response that would limit repeat dosing.

The kinetics and magnitude of the antibody response to any of the PAvariants disclosed herein can be determined as follows. For example, theantigenic response to PSA-PA1 (SEQ ID NO: 4) can be determined inimmunocompetent mice, to develop a dosing regimen that can be used in aimmunocompetent human. Immunocompetent mice (C57-BL6) are administerediv doses of PSA-PA1 (SEQ ID NO: 5) both daily×5 and weekly×3 at a doserange from 0.1 μg to 5 μg. Mice are sacrificed at varying intervals(e.g. following single dose, following multiple doses) and serumobtained. An ELISA-based assay can be used to detect presence ofanti-proaerolysin antibodies. In this assay, a defined quantity ofproaerolysin is fixed to the polystyrene surface in 96-well plates.Following adequate blocking with bovine serum albumin (BSA), serum frommice exposed to proaerolysin is added to the wells at varying dilutions.After a defined incubation time, wells are washed, and alkalinephosphatase linked goat-anti-mouse secondary antibody is added, followedby substrate. The amount of antibody present is determined by measuringabsorbance in a spectrophotometer, which permits determination of thetime course and magnitude of the antibody response by varying schedulesand doses of iv PSA-PA1.

To decrease antigenicity of proaerolysin, the native binding domain canbe functionally deleted and replaced, for example with LHRH, asdescribed in Example 6. The antigenicity of such peptides can bedetermined following exposure to varying schedules of LHRH-proaerolysinproteins which lack portions of the native binding domain using themethods described above. Another method that can be used to allowcontinued treatment with prostate-specific protease activated toxins isto use alternative lytic toxins with non-overlapping antigenicity (seeExample 10). One example is to use a modified, structurally relatedbacterial toxin such as Clostridium septicum alpha toxin that can alsobe activated by a prostate-specific protease, such as PSA, but would notbe recognized or neutralized by antibodies that recognize PA (seeExample 10). Another example is to use a pore-forming toxin produced byhuman tissues, such as human perforin produced by cytolytic human Tcells. Modified performs in which the wild type activation sequence isreplaced by peptides that are substrates for prostate-specificproteases, such as PSA, can be administered and not produce an antibodyresponse because the proteins are of human origin.

In addition, methods are provided to determine whether the antibodyproduced can neutralize the cytolytic properties of aerolysin. Adisclosed modified PA (such as SEQ ID NOS: 4, 24 and 25) is incubatedwith PSA to activate the toxin. Activated toxin is incubated withantibody containing serum at varying doses. Washed RBC's are added todetermine degree of hemolysis versus control (non-serum exposed toxin)as described above in Example 2.

In addition, PSA-producing tumor bearing animals can be inoculated twicewith PSA-activated proaerolysin toxins, then rechallenged with a lethaldose of toxin (see Example 4) to determine if antibody neutralizestoxicity in vivo. Subsequently, the anti-tumor response followingintratumoral injection of vaccinated animals is assessed, to determineif antibodies neutralize toxin when injected intratumorally.

Example 8 Induction of a Systemic Immunostimulatory Response

This example provides methods that can be used to demonstrate thatproaerolysin-mediated cell lysis produces a systemic immunostimulatoryeffect. Such a systematic immunostimulatory effect resulting from anintraprostatic administration of the toxins disclosed herein wouldprovide both local therapy for prostate cancer within the prostategland, while simultaneously induce a systemic antitumor effect againstoccult micrometastatic disease. Alternatively or in addition, subjectscan be vaccinated with modified proaerolysin-lysed prostate cancer cellsin the presence or absence of cytokines, such as GMCSF, to treatrecurrent or initial metastatic disease.

Administration of Prostate Tumor Cells Lysed with Modified Proaerolysin

To demonstrate that modified proaerolysin treated cells stimulate asystemic immune response in a subject, the following methods can beused. Briefly, subjects (such as immunocompetent mice or a human subjecthaving prostate cancer) are administered prostate tumor cells (such asTC2 mouse prostate tumor cells, prostate cancer cells obtained from thesubject having prostate cancer, and/or human prostate cancer cell linesfor administration to patients) which have been lysed with one or moremodified proaerolysin molecules disclosed herein. To determine whether asystemic immune response occurs, mice that have been administered lysedprostate cancer cells can be rechallenged with the same cells and growthof these inoculated tumor cells measured. For example, C57-BL6 mice aresubcutaneously injected with proaerolysin lysed TC2 cells. To accomplishthis, 10⁷ TC2 cells are lysed by incubation with proaerolysin for 1 hourat 37° C. in sterile phosphate buffered saline (PBS). Animals areadministered two weekly injections of this cell lysate to stimulate animmune response to the TC2 cells. Animals are subsequently rechallengedwith a subcutaneous injection of TC2 cancer cells one week after thesecond lysate inoculation. TC2 is a mouse prostate cancer cell linederived from cancerous tissue isolated from a TRAMP mouse (Foster etal., Cancer Res. 57:3325-30, 1997). Control subjects will receive asimilar number of cells that have been lysed by freezing and thawing, orthat have been treated with radiation to induce apoptosis. Anothercontrol group will receive only the modified proaerolysin peptide. Tumorgrowth is compared between the groups using the methods described inExample 5.

For human subjects, prostate cancer cells can be obtained from patientat time of prostatectomy or biopsy or human prostate cancer cell linescan be used. Examples of human prostate cancer cell lines includePSA-producing LNCaP (such as ATTC Nos. CRL-1740 and CRL-10995) or CW22R(ATCC No. CRL-2505 and Nagabhushan et al., Cancer Res. 56(13):3042-6,1996) cells, or PSA non-producing PC-3 (ATCC No. CRL-1435) and DU 145(ATCC No. HTB-81). Approximately 10⁷-10⁸ cells from either source(patient or cell lines) are incubated with 1 μg of PA (wild-type orvariant) for 1 hour at 37° C. in sterile PBS. The resulting lysate ismixed thoroughly using vigorous pipetting, and suspension isadministered to a subject via subcutaneous injection of ˜0.5 ml.Patients can be treated weekly for 3 doses. Patients are closelymonitored with weekly physical exams. Immune response to subcutaneous PAcan be monitored by assaying presence of antibodies to PSA, PSMA, andhK2, and assaying for B and T cell response using methodology previouslydescribed (Simons et al. Cancer Res. 59:5160-8, 1999).

Alternatively, or in addition, prostate cancer cells from a patient orprostate cancer cell lines engineered to express immunostimulatoryproteins (such as GM-CSF) are incubated with PA (wild-type or variant)and used to produce the lysate (for example see Simons et al. CancerRes. 59:5160-8, 1999). PA-lysed prostate cancer cells can beco-administered with irradiated prostate cancer cells producingimmunostimulatory molecules. Irradiation induces cells to undergoapoptosis. The combination of cells killed by cytolysis and cells killedby induction of apoptosis is expected to produce superiorimmunostimulatory effects (Sauter et al. J. Exp. Med. 191:423-33, 2000).In one example, a subject is co-administered PA-lysed prostate cancercells mixed with immunostimulatory protein. In another example, PA-lysedprostate cancer cells are administered subcutaneously, and animmunostimulatory protein, such as GM-CSF, interleukins, interferons,G-CSF (Dranoff et al. Proc. Natl. Acad. Sci. USA 90:3539-43, 1993) issystemically administered.

Intraprostatic Administration of Modified Proaerolysin

Another method that can be used to stimulate a systemic immune responsein a subject having prostate cancer is to administer the modifiedproaerolysin toxins disclosed herein directly into the prostate gland ofthe subject. For example, modified proaerolysin toxins can beadministered directly into the prostate gland (or the tumor itself) of ahuman subject having a prostate tumor, or into transgenic ProHA micefollowed by adoptive transfer of hemagglutinin (HA) primed T cells. Itis expected that cytolysis following intraprostatic injection ofmodified proaerolysin toxins will provoke immune response to HA in theprostate gland of ProHA mice, or to prostate tumor antigens in humans.ProHA transgenic mice express the influenza protein HA under the controlof the prostate-restricted promoter probasin. ProHA mice express HA onlyin the prostate gland. In this system, specific CD4 and CD8 T cells fromseparate donor mice are primed by exposure to HA and then adoptivelytransferred into the HA transgenic animal.

Subjects are administered a sublethal intraprostatic dose of modifiedproaerolysin (for instance determined using the methods described inExample 4). Because ProHA mice do not produce PSA or an enzymaticallyequivalent homolog, wild type aerolysin is used. However in humans, oneor more of the disclosed modified proaerolysin toxins are administered.Twenty-four hours after the intraprostatic injection, some mice aresacrificed and prostates removed and analyzed for degree of necrosis. Asecond group of injected mice will receive HA specific CD4 and CD8 Tcells, which have been harvested from TCR transgenic donors inoculatedwith 10⁹ pfu of recombinant hemagglutinin expressing vaccinia virus aspreviously described (Staveley-O'Carroll et al., Proc. Natl. Acad. Sci.USA 95:1178-83, 1998). These T cells are Thy 1.1+ and are transferredinto the ProHA mice which are Thy1.2+. Four days after adoptive transferinto the proaerolysin treated ProHA mice, recipient mice are sacrificed,and the spleen, axillary (irrelevant) and prostate draining nodesdissected, dissociated and washed.

To assess activation of specific T cells, the following assays can beperformed: One million isolated cells are stained using a Phycoerythrinlabeled anti Thy1.1 antibody and cychrome labeled antiCD4 or CD8antibody (Pharmingen, San Diego, Calif.). Isolated cells are analyzedusing FacsScan (Becton Dickinson). The index of expansion of HA specificT cells is calculated by dividing the percentage of clonotypic (Thy1.1+) cells in the ProHA treated animals by the percentage of clonotypiccells in an identical tissue location in untreated ProHA controls. Inaddition, the ratio of percentage of clonotypic cells in the prostatedraining versus irrelevant lymph nodes provides an index of prostatespecific proliferation following PA treatment. In a another assay,antigen-specific proliferation is measured as described previously(Adler et al. J. Exp. Med. 187:1555-64, 1998). Briefly, pooledlymphocytes or splenocytes are incubated with HA class II peptide (CD4cell specific proliferation) or HA class I peptide (CD8 cell specificproliferation). Cells are cultured for three days then pulsed overnightwith tritiated thymidine followed by harvest and determination ofcounts. Increased incorporated counts over control signifies specific Tcell activation.

The prostate glands from the PA treated ProHA mice that received HAspecific Thy1.1 T cells are analyzed as follows. Fixed prostate sectionsfrom treated mice are stained with biotinylated Thy1.1 antibody(Pharmingen) and counterstained with streptavidin peroxidase usingstandard immunohistochemical procedures. To determine extent ofstimulation, the degree of positive staining is compared to salinetreated controls.

To assess whether intraprostatic proaerolysin can effect subsequenttumor development, growth or pattern of spread, the following methodscan be used. Proaerolysin (or the modified proaerolysin peptidesdisclosed herein) can be injected into prostate of TRAMP mice at varioustime points in the life cycle of the animal (pre-pubescent, postpubescent prior to metastatic disease, with metastatic disease).Anesthetized animals are injected intraprostatically under sterileconditions. At various time points following intraprostatic inoculation,animals are sacrificed and necropsy performed to evaluate extent oftumor. Prostate glands are removed and immunohistochemical analysisperformed to assess presence of precursor lesions and overt prostatecancer. Similarly, pre-pubescent TRAMP animals can be twicesubcutaneously administered proaerolysin lysed TC2 cells or proaerolysinlysed primary tumors removed from late stage cancer bearing TRAMP mice.These animals are followed and sacrificed at defined time points toassess time course and magnitude of tumor development compared tocontrols.

A similar approach can be used to administer modified PA to patientswith localized prostate cancer. Such intraprostatic modified PA therapycan be used as initial treatment for localized prostate cancer eitheralone, or in combination with radiation (external beam or brachytherapy)and/or androgen ablation therapy. The intraprostatic modified PA therapycan also be administered to patients who have failed radiation therapyand are suspected to only have a local recurrence of prostate cancerwithin the prostate gland. The intraprostatic modified PA therapy canalso be given to patients with localized and metastatic prostate cancerto treat the localized cancer directly and to treat the metastaticcancer via stimulation of a systemic anti-tumor immune response.

To produce the intraprostatic modified PA therapy, modified PA isinjected into the prostate gland of patients according to a predefinedtemplate similar to that used to administer intraprostaticbrachytherapy. The techniques and equipment required for intraprostaticadministration are also similar to those used for brachytherapy and havebeen previously described (Deweese et al., Cancer Res. 61:7464-72,2001). To determine the appropriate dose to be administeredintraprostatically, a dose finding clinical trial is performed. Patientswill receive multiple injections (20-80) at predefined sites toencompass the entire prostate gland. The total dose of administeredmodified PA will range from about 0.1-1.0 mg, and not more than 10 mgtotal. The dose per injection will be determined by dividing total doseby total number of injections. Patients are treated as in patients andmonitored in the hospital for 48 hours post injection. Subsequently,patients will be examined weekly for signs of toxicity. MRI of theprostate can be used to monitor direct treatment effect on prostaticsize. Immune response to intraprostatic PA will be monitored aspreviously described (Simons et al. Cancer Res. 59:5160-8, 1999).

Example 9 Additional PSA Cleavage Sites

Additional PSA cleavage sites are known, based on the PSA-cleavage mapof human seminal proteins semenogelin I and II, and a cellulose membranebased assay (see Table 2 and Denmeade et al., Cancer Res., 57:4924-30,1997). The PSA-cleavage sites shown in Table 2 can substitute for thewild-type furin protease activation site of proaerolysin (amino acids427-432 of SEQ ID NO: 2), using the methods described in Example 1.Briefly, recombinant PCR can be used to replace the furin site of PAwith a PSA-specific cleavage site, such as those shown in Tables 1 and2. The variant PA sequence is subcloned into pMMB66HE for expression inE. coli. Recombinant clones are transferred into a protease deficientstrain of A. salmonicida, and the resulting variant proaerolysin proteinpurified by hydroxyapatite chromatography and ion exchangechromatography. TABLE 2 Kinetics of PSA hydrolysis.* PSA SubstrateK_(cat)/K_(m) (SEQ ID NO) K_(m) (uM) K_(cat) (S⁻¹) (s⁻¹M⁻¹) KGISSQY (15)160 0.043 270 SRKSQQY (16) 90 0.023 260 ATKSKQH (17) 1310 0.0091 6.9KGLSSQC (18) 300 0.0017 5.6 LGGSSQL (19) 900 0.0037 4.1 EHSSKLQ (20)1165 0.012 10.6 HSSKLQ  (5) 470 0.011 23.6 SKLQ    (21) 813 0.020 24.6*Peptides were fluorescently labeled (aminomethyl coumarin). Assays wereperformed in 50 mM Tris, 0.1 M NaCl, pH 7.8.

The sequences shown in Table 2 include substrates that were efficientlybut not specifically hydrolyzed by PSA (KGISSQY; SEQ ID NO: 15) andSRKSQQY; SEQ ID NO: 16), and those that were not efficient, but werespecifically hydrolyzed by PSA (ATKSKQH; SEQ ID NO: 17 and LGGSSQL; SEQID NO: 19). The characteristics of these modified toxins can be comparedto PSA-PA1 containing the HSSKLQ (SEQ ID NO: 5) sequence. As a control,a modified toxin is produced in which the activation site is completelydeleted (EX-PA).

These purified toxins are screened for PSA hydrolysis using thehemolysis assay described in Example 3. Wild type proaerolysin is notactivated or cytolytic to erythrocytes. Activation of proaerolysin byproteases, however, results in rapid hemolysis. Briefly, to assay PSAactivation, varying concentrations of purified variant PA toxins areincubated with PSA for 1 hour, washed, and serum free red blood cells(RBC's) (2% v/v) added. After another 1 hour, incubation mixtures arecentrifuged and absorbance of the supernatant at 540 nm determined.Minimal concentration of modified toxin required to lyse 50% of RBC's isdetermined to rank toxins on basis of efficiency of PSA hydrolysis.

To determine both PSA-activation and specificity of activation,cytotoxicity assays are performed by exposing purified PSA-proaerolysintoxins to PSA-producing (LNCaP) and non-producing cancer cell lines(TSU) in vitro, using the methods described in Example 2. Theconcentration required to kill 50% of cells (IC₅₀) can be determined foreach variant PA toxin against both LNCaP and TSU lines. Thefold-difference in cytotoxicity is used to rank PSA-activated toxins onbasis of specificity.

Variant PA toxins can also be screened in vivo for antitumoral activityby intratumoral injection into PSA-producing LNCaP xenografts in nudemice, using the methods described in Example 5. As described above inExample 5, intratumoral injection of the PSA-PA1 toxin reduces the tumorwithin 48 hours. In contrast, wild type proaerolysin, which is moreefficiently activated by both PSA-producing and non-producing cell linesin vitro, had minimal effect against the LNCaP xenografts. Thisindicates that the kinetics of toxin activation may be important in theoverall antitumor effect. PSA-PA1 is activated more specifically by PSA,but the kinetics of activation are slower than the wild type toxin. Thismay allow PSA-PA1 to distribute more widely throughout the tumor.Therefore, better PSA substrates in the activation site, paradoxicallymay result in decreased overall antitumor effect in vivo.

To assess distribution of PSA-activated proaerolysin versus wild type PAintratumorally or into normal tissue, fluorescently labeled (such asFITC) PSA-PA1 and PA proteins can be used. PSA-producing LNCAPxenografts are injected with the fluorescently labeled PSA-PA1 and PA.At 24 hours following intratumoral injection, tumors are harvested,fixed and sectioned for microscopic analyses. Fluorescently labeledproaerolysin proteins that have inserted into cell membranes areretained throughout the fixation process. These slides are then analyzedusing a fluorescence microscope equipped with the appropriate filter setto determine degree of distribution of the proaerolysin toxinsthroughout the tumor specimen.

Subsequently, variant PA toxins are injected into LNCaP xenografts andantitumor response assessed after 48 hours by tumor measurement, usingthe methods described in Example 5. The variant PA toxins are ranked onthe basis of in vivo antitumor effect following intratumoral injection.

In addition, variant PA toxins can be tested for overall systemictoxicity by determining the dose that kills 100% of mice (i.e. LD₁₀₀)following a single intravenous injection, using the methods described inExample 4.

Using these methods, PSA-activated variant PA toxins which are mostefficiently and specifically activated by PSA, are the least toxicsystemically and produce the most pronounced antitumor effect in vivo,are identified. Such PSA-activated variant PA toxins can also bemodified using the methods described in Example 6.

Example 10 Reduction of Antigenicity of PSA-Activated ModifiedProaerolysin Toxin

This example describes additional methods than can be used to produceand characterize antigenicity and anti-tumor efficacy of other modifiedpore forming protease activated protoxins.

One method to overcome the potential antigenicity of the disclosedvariant PA toxins is to sequentially administer structurally relatedprotoxins that are similarly activated by PSA, but which are notrecognized by proaerolysin antibodies. Examples of such protoxinsinclude, but are not limited to, Clostridium septicum alpha toxin(Ballard et al., Infect. Immun. 63:340-4, 1995; Gordon et al. J. Biol.Chem. 274:27274-80, 1999; Genbank Accession No. S75954), BacillusThuringiensis delta-toxin (Genbank Accession No. D00117), and humanperforin (Genbank Accession No. NM005041). While mechanistically similarto aerolysin, these protoxins have different peptide sequences such thatantibodies specific to proaerolysin would not recognize them. Theseprotoxins have been cloned and recombinant forms produced (Imagawa etal., FEMS. Microbiol. Lett. 17:287-92, 1994; Meza et al. FEMS Microbiol.Lett. 145:333-9, 1996).

These protoxins, like proaerolysin, contain a C-terminal inhibitorypeptide that must be removed by proteolytic cleavage for activation tooccur. The activation site within each of these protein toxins has beendefined. For Clostridium septicum alpha toxin, the activation site is afurin cleavage site (Gordon et al., Infect. Immun. 65:4130-4, 1997). Theactivation site of Bacillus Thuringiensis delta-toxin is cleaved byproteases in the midgut of certain insects (Miranda et al., InsectBiochem. Mol. Biol. 31:1155-63, 2001). For human perforin, theactivation sequence has been defined but the activating protease has notyet been identified (Uellner et al., EMBO J. 16:7287-96, 1997).

The activation site of each of these protoxins can be modified tocontain a prostate-specific protease cleavage site (such as the PSAcleavage sites shown in Tables 1-2) using the methods described inExample 1. If desired, the native protoxin binding domain can befunctionally deleted and replaced with a prostate-tissue specificbinding domain, using the methods described in Example 6. Alternatively,the activation site is not modified, but the binding domain isfunctionally deleted and replaced with a prostate-tissue specificbinding domain, using the methods described in Example 6. These modifiedprotoxins are assayed for protease activation, for example using the RBChemolysis assay (Example 3), for in vitro activity against PSA-producingand non-PSA producing cell lines (Example 2) and stability in humanserum (Example 3). If these toxins are specifically and efficientlyactivated by PSA, then they are tested in vivo for activity againstPSA-producing xenografts using the methods described in Example 5. Inaddition, kinetics and magnitude of antibody production will bedetermined using methods described in Example 7. Serum from animalstreated with each toxin is screened for cross reactivity with each ofthe other members of the toxin family to determine degree ofcross-recognition.

Another method for reducing the systemic immune response is toadminister immunosuppressive therapies. Examples of immunosuppressivetherapies include, but are not limited to, systemic or topicalcorticosteroids (Suga et al., Ann. Thorac. Surg. 73:1092-7, 2002),cyclosporin A (Fang et al., Hum. Gene Ther. 6:1039-44, 1995),cyclophosphamide (Smith et al., Gene Ther. 3:496-502, 1996),deoxyspergualin (Kaplan et al., Hum. Gene Ther. 8:1095-1104, 1997) andantibodies to T and/or B cells [e.g. anti-CD40 ligand, anti CD4antibodies, anti-CD20 antibody (Rituximab)] (Manning et al., Hum. GeneTher. 9:477-85, 1998). Such agents can be administered before, during,or subsequent to administration of modified PA molecules and/or celllysates produced by incubation with PA (wild-type or variant).

Example 11 Production of Sequence Variants

Disclosed herein are agents and methods for treating prostate cancer byadministration of a modified proaerolysin peptide which includes aprostate-specific protease cleavage site. It is understood by thoseskilled in the art that use of other proaerolysin, PSA, LHRH, and PSMAsequences (such as polymorphisms, fragments, or variants) can be used topractice the methods of the present disclosure, as long as thedistinctive functional characteristics of the sequence are retained. Forexample, proaerolysin variants can be used to practice the methodsdisclosed herein if they retain their ability to be activated by aprostate-specific protease and form pores in cell membranes, resultingin cell death. This activity can readily be determined using the assaysdisclosed herein, for example those described in EXAMPLES 2-5. In yetother embodiments, modified proaerolysin molecules have thecharacteristic of specifically lysing PSA-producing cells (for example,lyse PSA-producing cells to a greater extent than non-PSA producingcells).

This disclosure facilitates the use of DNA molecules, and therebyproteins, derived from a native protein but which vary in their precisenucleotide or amino acid sequence from the native sequence. Suchvariants can be obtained through standard molecular biology laboratorytechniques and the sequence information disclosed herein.

DNA molecules and nucleotide sequences derived from a native DNAmolecule can also be defined as DNA sequences which hybridize understringent conditions to the DNA sequences disclosed, or fragmentsthereof. Hybridization conditions resulting in particular degrees ofstringency vary depending upon the nature of the hybridization methodand the composition and length of the hybridizing DNA used. Generally,the temperature of hybridization and the ionic strength (especially theNa⁺ concentration) of the hybridization buffer determines hybridizationstringency. Calculations regarding hybridization conditions required forattaining particular amounts of stringency are discussed by Sambrook etal. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,1989, Chapters 9 and 11), herein incorporated by reference.Hybridization with a target probe labeled with [³²P]-dCTP is generallycarried out in a solution of high ionic strength such as 6×SSC at atemperature that is about 5-25° C. below the melting temperature, T_(m).An example of stringent conditions is a salt concentration of at leastabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3 and a temperature of at least about 30° C. for short probes (e.g. 10to 50 nucleotides). Stringent conditions can also be achieved with theaddition of destabilizing agents such as formamide. For example,conditions of 5×SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH7.4) at 25-30° C. are suitable for allele-specific probe hybridizations.

The degeneracy of the genetic code further widens the scope of thepresent disclosure as it enables major variations in the nucleotidesequence of a DNA molecule while maintaining the amino acid sequence ofthe encoded protein. For example, the amino acid Ala is encoded by thenucleotide codon triplet GCT, GCG, GCC and GCA. Thus, the nucleotidesequence could be changed without affecting the amino acid compositionof the encoded protein or the characteristics of the protein. Based uponthe degeneracy of the genetic code, variant DNA molecules may be derivedfrom a cDNA molecule using standard DNA mutagenesis techniques asdescribed above, or by synthesis of DNA sequences. DNA sequences whichdo not hybridize under stringent conditions to the cDNA sequencesdisclosed by virtue of sequence variation based on the degeneracy of thegenetic code are also comprehended by this disclosure.

PA variants, fragments, fusions, and polymorphisms will retain theability to lyse PSA-producing cells, as determined using the assaysdisclosed herein (for example, see Examples 2 and 5). Variants andfragments of a the disclosed sequences retain at least 70%, 80%, 85%,90%, 95%, 98%, or greater sequence identity to a protein amino acidsequence and maintain the functional activity of the protein asunderstood by those in skilled in the art.

Example 12 Recombinant Expression of Proteins

With publicly available cDNA and corresponding amino acid sequences, aswell as the disclosure herein of PA variants, fragments and fusions, theexpression and purification of any protein by standard laboratorytechniques is enabled. The purified protein can be used for patienttherapy. One skilled in the art will understand that the disclosedmodified PA toxins can be produced in any cell or organism of interest,and purified prior to administration to a subject.

Methods for producing recombinant proteins are well known in the art.Therefore, the scope of this disclosure includes recombinant expressionof any protein. For example, see U.S. Pat. No. 5,342,764 to Johnson etal.; U.S. Pat. No. 5,846,819 to Pausch et al.; U.S. Pat. No. 5,876,969to Fleer et al. and Sambrook et al. (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., 1989, Ch. 17, herein incorporated byreference).

Example 13 Peptide Modifications

Modified PA proteins which are activated by a prostate-specificprotease, such as PSA, can be modified using a variety of chemicaltechniques to produce derivatives having essentially the same activityas the unmodified peptides, and optionally having other desirableproperties (such as reduced antigenicity). For example, carboxylic acidgroups of the peptide, whether carboxyl-terminal or side chain, can beprovided in the form of a salt of a pharmaceutically-acceptable cationor esterified to form a C₁-C₁₆ ester, or converted to an amide offormula NR₁R₂ wherein R₁ and R₂ are each independently H or C₁-C₁₆alkyl, or combined to form a heterocyclic ring, such as a 5- or6-membered ring. Amino groups of the peptide, whether amino-terminal orside chain, can be in the form of a pharmaceutically-acceptable acidaddition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic,maleic, tartaric and other organic salts, or may be modified to C₁-C₁₆alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chain can be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chain can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups. Those skilled in the art will also recognize methods forintroducing cyclic structures into the peptides disclosed herein toselect and provide conformational constraints to the structure thatresult in enhanced stability. For example, a carboxyl-terminal oramino-terminal cysteine residue can be added to the peptide, so thatwhen oxidized the peptide will contain a disulfide bond, generating acyclic peptide. Other peptide cyclizing methods include the formation ofthioethers and carboxyl- and amino-terminal amides and esters.

To maintain a functional peptide, particular peptide variants willdiffer by only a small number of amino acids from a peptide. Suchvariants can have deletions (for example of 1-3 or more amino acids),insertions (for example of 1-3 or more residues), or substitutions thatdo not interfere with the desired activity of the peptide.Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. In particular embodiments, such variants have amino acidsubstitutions of single residues, for example 1, 3, 5 or even 10substitutions in a protein.

Peptidomimetic and organomimetic embodiments are also disclosed herein,whereby the three-dimensional arrangement of the chemical constituentsof such peptido- and organomimetics mimic the three-dimensionalarrangement of the peptide backbone and component amino acid sidechainsin the peptide, resulting in such peptido- and organomimetics of amodified PA toxin which has the ability to lyse PSA-producing cells. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology (ed. Munson, 1995), chapter 102 for adescription of techniques used in CADD.

Example 14 Methods for Expressing Modified Proaerolysin Peptides

As an alternative to (or in addition to) administration of a modifiedproaerolysin peptide to treat prostate cancer, long term or systemictreatment (for example to treat or prevent metastasis of the tumor) canbe achieved by expressing the disclosed modified proaerolysin toxins invivo.

The present disclosure provides methods of expressing a modifiedproaerolysin peptide in a cell or tissue in vivo. In one example,transfection of the cell or tissue occurs in vitro. In this example, thecell or tissue (such as a graft) is removed from a subject and thentransfected with an expression vector containing a cDNA encoding theprotein of interest. The transfected cells will produce functionalprotein and can be reintroduced into the subject. In another example, anucleic acid encoding the protein of interest is administered to asubject directly (such as intravenous, intratumor or intraprostate), andtransfection occurs in vivo.

The modified proaerolysin peptides and methods disclosed herein can beused to treat a subject with a prostate tumor. Such a method woulddecrease the volume of the tumor, and in some embodiments, prevent ortreat metastasis of the prostate tumor.

The scientific and medical procedures required for human celltransfection are now routine. The public availability of proaerolysin,binding domains, and prostate-specific protease protein and cDNAsequences allows the development of human (and other mammals) in vivogene expression based upon these procedures. In addition, particularvariant proaerolysin molecules are disclosed herein. Immunotherapy ofmelanoma patients using genetically engineered tumor-infiltratinglymphocytes (TILs) has been reported by Rosenberg et al. (N. Engl. J.Med. 323:570-8, 1990). In that study, a retrovirus vector was used tointroduce a gene for neomycin resistance into TILs. A similar approachcan be used to introduce a modified proaerolysin peptide cDNA into asubject having a prostate cancer.

A general strategy for transferring genes into donor cells is disclosedin U.S. Pat. No. 5,529,774, incorporated by reference. Generally, a geneencoding a protein having therapeutically desired effects is cloned intoa viral expression vector, and that vector is then introduced into thetarget organism. The virus infects the cells, and produces the proteinsequence in vivo, where it has its desired therapeutic effect (Zabner etal. Cell 75:207-16, 1993).

It may only be necessary to introduce the DNA or protein elements intocertain cells or tissues. For example, if a subject only has a prostatetumor, introducing the protein or DNA into only the prostate (or tumor)may be sufficient. However, in some instances, it may be moretherapeutically effective and simple to treat all of a subject's cells,or more broadly disseminate the vector, for example by intravascular(i.v.) or oral administration. For example, if a subject has a prostatetumor that has metastasized, introducing the protein or DNA systemicallymay be necessary.

The nucleic acid sequence encoding at least one therapeutic agent, suchas a modified proaerolysin toxin, is under the control of a suitablepromoter. Suitable promoters which can be used include, but are notlimited to, the gene's native promoter, retroviral LTR promoter, oradenoviral promoters, such as the adenoviral major late promoter; theCMV promoter; the RSV promoter; inducible promoters, such as the MMTVpromoter; the metallothionein promoter; heat shock promoters; thealbumin promoter; the histone promoter; the α-actin promoter; TKpromoters; B19 parvovirus promoters; and the ApoAI promoter. In oneexample, the promoter is a prostate-specific promoter, such as aprobasin promoter. However the disclosure is not limited to specificforeign genes or promoters.

The recombinant nucleic acid can be administered to the subject by anymethod which allows the recombinant nucleic acid to reach theappropriate cells. These methods include injection, infusion,deposition, implantation, or topical administration. Injections can beintradermal or subcutaneous. The recombinant nucleic acid can bedelivered as part of a viral vector, such as avipox viruses, recombinantvaccinia virus, replication-deficient adenovirus strains or poliovirus,or as a non-infectious form such as naked DNA or liposome encapsulatedDNA, as further described in EXAMPLE 15.

Example 15 Viral Vectors for in vivo Gene Expression

Adenoviral vectors include essentially the complete adenoviral genome(Shenk et al., Curr. Top. Microbiol. Immunol. 111:1-39, 1984).Alternatively, the adenoviral vector is a modified adenoviral vector inwhich at least a portion of the adenoviral genome has been deleted. Inone example, the vector includes an adenoviral 5′ ITR; an adenoviral 3′ITR; an adenoviral encapsidation signal; a DNA sequence encoding atherapeutic agent; and a promoter for expressing the DNA sequenceencoding a therapeutic agent. The vector is free of at least themajority of adenoviral E1 and E3 DNA sequences, but is not necessarilyfree of all of the E2 and E4 DNA sequences, and DNA sequences encodingadenoviral proteins transcribed by the adenoviral major late promoter.In another example, the vector is an adeno-associated virus (AAV) suchas described in U.S. Pat. No. 4,797,368 (Carter et al.) and inMcLaughlin et al. (J. Virol. 62:1963-73, 1988) and AAV type 4 (Chioriniet al. J. Virol. 71:6823-33, 1997) and AAV type 5 (Chiorini et al. J.Virol. 73:1309-19, 1999).

Such a vector can be constructed according to standard techniques, usinga shuttle plasmid which contains, beginning at the 5′ end, an adenoviral5′ ITR, an adenoviral encapsidation signal, and an E1a enhancersequence; a promoter (which may be an adenoviral promoter or a foreignpromoter); a tripartite leader sequence, a multiple cloning site (whichmay be as herein described); a poly A signal; and a DNA segment whichcorresponds to a segment of the adenoviral genome. The DNA segmentserves as a substrate for homologous recombination with a modified ormutated adenovirus, and may encompass, for example, a segment of theadenovirus 5′ genome no longer than from base 3329 to base 6246. Theplasmid can also include a selectable marker and an origin ofreplication. The origin of replication may be a bacterial origin ofreplication. A desired DNA sequence encoding a therapeutic agent can beinserted into the multiple cloning site of the plasmid.

Examples of vectors which can be used to practice the methods disclosedherein include, but are not limited to, those disclosed in: WO 95/27512to Woo et al.; WO 01/127303 to Walsh et al.; U.S. Pat. No. 6,221,349 toCouto et al.; U.S. Pat. No. 6,093,392 to High et al.

Example 16 Generation and Expression of Fusion Proteins

Methods for making fusion proteins are well known to those skilled inthe art. For example U.S. Pat. No. 6,057,133 to Bauer et al. (hereinincorporated by reference) discloses methods for making fusion moleculescomposed of human interleukin-3 (hIL-3) variant or mutant proteinsfunctionally joined to a second colony stimulating factor, cytokine,lymphokine, interleukin, hematopoietic growth factor or IL-3 variant.U.S. Pat. No. 6,072,041 to Davis et al. (herein incorporated byreference) discloses the generation of fusion proteins comprising asingle chain Fv molecule directed against a transcytotic receptorcovalently linked to a therapeutic protein.

Similar methods can be used to generate fusion proteins comprising PA(or variants, fragments, etc. thereof) linked to other amino acidsequences, such as a prostate specific binding domain (for example LHRHor an antibody). Linker regions can be used to space the two portions ofthe protein from each other and to provide flexibility between them. Thelinker region is generally a polypeptide of between 1 and 500 aminoacids in length, for example less than 30 amino acids in length. Thelinker joining the two molecules can be designed to (1) allow the twomolecules to fold and act independently of each other, (2) not have apropensity for developing an ordered secondary structure which couldinterfere with the functional domains of the two proteins, (3) haveminimal hydrophobic or charged characteristic which could interact withthe functional protein domains and (4) provide steric separation of thetwo regions. Typically surface amino acids in flexible protein regionsinclude Gly, Asn and Ser. Other neutral amino acids, such as Thr andAla, can also be used in the linker sequence. Additional amino acids canbe included in the linker due to the addition of unique restrictionsites in the linker sequence to facilitate construction of the fusions.Other moieties can also be included, as desired. These can include abinding region, such as avidin or an epitope, such as a polyhistadinetag, which can be useful for purification and processing of the fusionprotein. In addition, detectable markers can be attached to the fusionprotein, so that the traffic of the fusion protein through a body orcell can be monitored conveniently. Such markers include radionuclides,enzymes, fluorophores, and the like.

Fusing of the nucleic acid sequences of PA (or variant, fragment etc.thereof), with the nucleic acid sequence of another protein (or variant,fragment etc. thereof), can be accomplished by the use of intermediatevectors. Alternatively, one gene can be cloned directly into a vectorcontaining the other gene. Linkers and adapters can be used for joiningthe nucleic acid sequences, as well as replacing lost sequences, where arestriction site was internal to the region of interest. Geneticmaterial (DNA) encoding one polypeptide, peptide linker, and the otherpolypeptide is inserted into a suitable expression vector which is usedto transform prokaryotic or eukaryotic cells, for example bacteria,yeast, insect cells or mammalian cells. The transformed organism isgrown and the protein isolated by standard techniques, for example byusing a detectable marker such as nickel-chelate affinitychromatography, if a polyhistadine tag is used. The resulting product istherefore a new protein, a fusion protein, which has modified PA joinedby a linker region to a second protein. To confirm that the fusionprotein is expressed, the purified protein is subjected toelectrophoresis in SDS-polyacrylamide gels, and transferred ontonitrocellulose membrane filters using established methods. The proteinproducts can be identified by Western blot analysis using antibodiesdirected against the individual components, i.e., polyhistadine tag andPA.

Example 17 Pharmaceutical Compositions and Modes of Administration

The pharmaceutically effective carriers useful herein are conventional.Remington's Pharmaceutical Sciences, by Martin, Mack Publishing Co.,Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery.

Administration of Peptides

In an embodiment in which a modified PA toxin, such as SEQ ID NOS: 4, 24and 25, is administered to a subject, the protein is delivered by anyroute used by those in the art. Examples include, but are not limitedto: intravenously, intratumorally, orally, intraprostatically,intramuscularly, subcutaneous injection, transdermal, etc. The presentdisclosure also provides pharmaceutical compositions which include atherapeutically effective amount of a modified PA toxin alone or with apharmaceutically acceptable carrier. Furthermore, the pharmaceuticalcompositions or methods of treatment can be administered in combination(or separately) with one or more other therapeutic treatments. Examplesof other therapeutics include, but are not limited to anti-tumor agents,cell lysates (such as those generated by incubation with a modified PAtoxin), non-lysed cells (such as those that have been killed byradiation), immunosuppressants (such as Rituximab, steroids), and/orcytokines (such as GM-CSF). Embodiments of the disclosure includingmedicaments can be prepared with conventional pharmaceuticallyacceptable carriers, adjuvants and counterions as would be known tothose of skill in the art.

The modified PA toxin can be administered in combination with at leastone, for example one or more pharmaceutically effective carriers, suchas a pharmaceutically and physiologically acceptable fluid. Examples ofpharmaceutically effective carriers include, but are not limited towater, physiological saline, balanced salt solutions, aqueous dextrose,sesame oil, glycerol, ethanol, combinations thereof, or the like, as avehicle. The carrier and composition can be sterile, and the formulationsuits the mode of administration. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

The composition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. For solidcompositions (e.g., powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, sodium saccharine,cellulose, magnesium carbonate, or magnesium stearate. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides.

The amount of modified PA toxin, such as SEQ ID NOS: 4, 24 and 25,effective in the treatment of a particular disorder or condition, suchas prostate cancer, will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. Inaddition, in vitro assays can be employed to identify optimal dosageranges (see Examples 2, 4, and 5). The precise dose to be employed inthe formulation will also depend on the seriousness of the disease ordisorder, and should be decided according to the judgment of thepractitioner and each subject's circumstances. Effective doses can beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. Examples of effective iv doses of a modified PAtoxin for a 70 kg human are about 1-10 mg of modified PA toxin, forexample about 1-5 mg, for example about 1-3 mg, for example about 2.8mg. Examples of effective intraprostatic or intratumor doses of amodified PA toxin for a 70 kg human are about 10-100 mg of modified PAtoxin, for example about 10-50 mg, for example about 10-30 mg, forexample about 28 mg.

The disclosure also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. Instructions for useof the composition can also be included.

Administration of Nucleic Acid Molecules

In an example in which a nucleic acid is employed to allow expression ofa nucleic acid in a cell, the nucleic acid is delivered intracellularly(e.g., by expression from a nucleic acid vector or by receptor-mediatedmechanisms). In one embodiment, a nucleic acid encodes for an modifiedPA, such as a SEQ ID NO: 4, 24 or 25.

Various delivery systems for administering a nucleic acid are known, andinclude encapsulation in liposomes, microparticles, microcapsules,receptor-mediated endocytosis (Wu and Wu, J. Biol. Chem. 1987,262:4429-32), and construction of therapeutic nucleic acids as part of aretroviral or other vector. Methods of introduction include, but are notlimited to, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, and oral routes. The compounds may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal, vaginal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local.

Liposomes fuse with the target site and deliver the contents of thelumen intracellularly. The liposomes are maintained in contact with thetarget cells for a sufficient time for fusion to occur, using variousmeans to maintain contact, such as isolation and binding agents.Liposomes can be prepared with purified proteins or peptides thatmediate fusion of membranes, such as Sendai virus or influenza virus.The lipids may be any useful combination of known liposome forminglipids, including cationic lipids, such as phosphatidylcholine. Otherpotential lipids include neutral lipids, such as cholesterol,phosphatidyl serine, phosphatidyl glycerol, and the like. For preparingthe liposomes, the procedure described by Kato et al. (J. Biol. Chem.1991, 266:3361) can be used.

Where the therapeutic molecule is a nucleic acid, administration can beachieved by an appropriate nucleic acid expression vector which isadministered so that it becomes intracellular, e.g., by use of aretroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see e.g.,Joliot et al., Proc. Natl. Acad. Sci. USA 1991, 88:1864-8), etc.Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination.

The vector pcDNA, is an example of a method of introducing the foreigncDNA into a cell under the control of a strong viral promoter (CMV) todrive the expression. However, other vectors can be used (see Example15). Other retroviral vectors (such as pRETRO-ON, Clontech), also usethis promoter but have the advantages of entering cells without anytransfection aid, integrating into the genome of target cells only whenthe target cell is dividing (as cancer cells do, especially during firstremissions after chemotherapy) and they are regulated. It is alsopossible to turn on the expression of a nucleic acid by administeringtetracycline when these plasmids are used.

Other plasmid vectors, such as pMAM-neo (Clontech) or pMSG (Pharmacia)use the MMTV-LTR promoter (which can be regulated with steroids) or theSV10 late promoter (pSVL, Pharmacia) or metallothionein-responsivepromoter (pBPV, Pharmacia) and other viral vectors, includingretroviruses. Examples of other viral vectors include adenovirus, AAV(adeno-associated virus), recombinant HSV, poxviruses (vaccinia) andrecombinant lentivirus (such as HIV). These vectors achieve the basicgoal of delivering into the target cell the cDNA sequence and controlelements needed for transcription. The present disclosure includes allforms of nucleic acid delivery, including synthetic oligos, naked DNA,plasmid and viral, integrated into the genome or not.

Having illustrated and described novel modified proaerolysin toxins, andmethods of using these molecules for treating prostate cancer andmetastases, it should be apparent to one skilled in the art that thedisclosure can be modified in arrangement and detail without departingfrom such principles. In view of the many possible embodiments to whichthe principles of our disclosure may be applied, it should be recognizedthat the illustrated embodiments are only particular examples of thedisclosure and should not be taken as a limitation on the scope of thedisclosure. Rather, the scope of the disclosure is in accord with thefollowing claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

1. A purified peptide comprising a variant proaerolysin amino acidsequence, wherein the variation of the proaerolysin amino acid sequencecomprises replacement of the furin cleavage site corresponding to thesequence of amino acids at positions 427-432 of SEQ ID NO: 2 by aprostate-specific protease cleavage site, wherein administration of 2.5to 25 μg of the peptide to a mouse having a prostate tumor xenograftreduces the proliferative index of the tumor to less than 1%.
 2. Thepeptide of claim 1, wherein the prostate-specific protease cleavage sitecomprises a prostate-specific antigen (PSA) cleavage site, a prostatespecific membrane antigen (PSMA) cleavage site, or a human glandularkallikrein 2 (hK2) cleavage site.
 3. The peptide of claim 2, wherein thePSA cleavage site comprises SEQ ID NO: 5, 8, 11, 14, 15, 16, 17, 18, 19,20, or
 21. 4. The peptide of claim 3, wherein the PSA cleavage sitecomprises SEQ ID NO:
 5. 5. A purified nucleic acid molecule encoding thepeptide of claim
 1. 6. The purified nucleic acid molecule of claim 5,wherein the nucleic acid molecule comprises the nucleic acid sequence ofSEQ ID NO:
 3. 7. A method for treating prostate cancer in a subject,comprising contacting prostate cancer cells of the subject with thepeptide of claim
 1. 8. The method of claim 7, wherein contactingprostate cancer cells of the subject with the peptide comprisesadministering the peptide to the subject.
 9. The method of claim 8,wherein the peptide is administered intratumorally and/orintraprostatically.
 10. The method of claim 8, wherein the peptide isadministered intravenously, intramuscularly, subcutaneously, or orally.11. The method of claim 7, wherein contacting prostate cancer cells ofthe subject with the peptide comprises: removing prostate cancer cellsfrom the subject; contacting the cells with the peptide, therebygenerating a cell lysate; and administering the cell lysate to thesubject.
 12. The method of claim 11, wherein the subject has ametastatic prostate tumor.
 13. The method of claim 11, furthercomprising administering GM-CSF to the subject.
 14. The method of claim13, further comprising administering irradiated prostate cancer cells tothe subject.
 15. The method of claim 7, wherein administration resultsin a reduction in prostate tumor cell volume.
 16. The method of claim 7,wherein administration results in a reduction of a metastatic prostatetumor.
 17. The method of claim 12, wherein administration results intreatment of the metastatic prostate tumor.
 18. A purified peptidecomprising a variant proaerolysin amino acid sequence, wherein thevariation of the proaerolysin amino acid sequence comprises afunctionally deleted proaerolysin binding domain corresponding to thesequence of amino acids at positions 1-83 of SEQ ID NO: 2 replaced witha prostate-tissue specific binding domain.
 19. The peptide of claim 18,wherein the prostate-tissue specific binding domain comprises a PSAantibody, an hK2 antibody, a PSMA antibody, a LHRH antibody, an LHRHsequence, a peptide sequence that binds PSMA, or any peptide sequencethat selectively binds to prostate cancer cells.
 20. A purified peptidecomprising a variant human perforin amino acid sequence, wherein thevariation of the human perforin amino acid sequence comprisesreplacement of the wild type perforin activation site by aprostate-specific protease cleavage site.